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B    3    tit. 2    3E3 


DIOXIDE  PRODUCTION  FROM 

:BRES  WHEN  RESTING 

HEN  STIMULATED 


A  DISSERTATION 

SUBMITTED   TO   THE   FACULTY  OF   THE   OGDEN   GRADUATE   SCHOOL 

OF   SCIENCE  IN  CANDIDACY  FOR  THE  DEGREE 

OF  DOCTOR   OF   PHILOSOPHY 

(DEPARTMENT  OF  PHYSIOLOGY) 


BY 
SHIRO  TASHIRO 


CHICAGO 

1912 


MEDICAL 

LHEHRAIEY 


tlbe  mntx>ersit£  of 


CARBON  DIOXIDE  PRODUCTION  FROM 

NERVE  FIBRES  WHEN  RESTING 

AND  WHEN  STIMULATED 


A  DISSERTATION 

SUBMITTED    TO    THE   FACULTY   OF    THE    OGDEN   GRADUATE    SCHOOL 

OF    SCIENCE   IN   CANDIDACY   FOR   THE   DEGREE 

OF  DOCTOR   OF   PHILOSOPHY 

(DEPARTMENT  OF  PHYSIOLOGY) 


BY 
SHIRO  /TASHIRQ 


/2 


CHICAGO 

IQI2 


Reprinted  from  the  American  Journal  of  Physiology 
Vol.  XXXII  — June  2,  1913  — No.  II 


CARBON  DIOXIDE  PRODUCTION  FROM  NERVE  FIBRES 
WHEN  RESTING  AND  WHEN  STIMULATED;  A 
CONTRIBUTION  TO  THE  CHEMICAL  BASIS  OF 
IRRITABILITY.1 

BY  SHIRO  TASHIRO 

[From  the  Department  of  Biochemistry  and  Pharmacology,  the  University  of  Chicago,  and  the 
Marine  Biological  Laboratory,  Woods  Hole,  Mass.] 

INTRODUCTION 

^  I  ^HERE  have  been  two  theories  of  the  nature  of  conduction  — • 
A      one  upheld  among  others  by  Hermann,  that  it  was  a  prop- 
agated chemical  change;   the  other,  at  present  the  dominant  view, 
that  it  is  a  propagated  physical  change. 

In  1901  Professor  Mathews  suggested  2  that  it  was  in  the  nature 
of  a  coagulative  wave  propagated  along  the  fibre;  this  coagulation  of 
the  nerve  colloids  leading  either  directly  or  indirectly  to  the  electrical 
disturbance  accompanying  the  impulse.  At  the  time,  there  was  no 
evidence  of  chemical  change  in  the  nerve  fibre,  and  its  indefatiga- 
bility  seemed  to  point  to  an  absence  of  metabolism.  Certain  facts 
were  known,  however,  which  were  difficult  to  reconcile  with  this  phys- 
ical theory.  Darwin  had  observed  that  in  Drosera,3  conduction 
occurred  only  if  the  protoplasm  had  oxygen;  and  Mathews  4  observed 
that  salts  would  not  stimulate  a  nerve,  or,  at  any  rate,  their  power  of 
stimulation  was  much  reduced  if  the  nerve  remained  in  the  body  for 
a  time  after  death,  or  if  the  nerve  were  brought  into  the  salt  solution 
in  an  atmosphere  of  hydrogen.  This  clearly  indicated  a  dependence 
of\he  irritability  on  oxygen. 

1  The  preliminary  report  of  these  investigations  was  given  in  part  in  Bio- 
chemical section  of  Eighth  International  Congress  for  applied  chemistry,  Sep- 
tember, 1912.     See  original  communications,  Eighth  International  Congress  of 
applied  chemistry,  xxvi,  p.  163.     See  also  this  Journal  1913,  xxxi,  p.  xxii. 

2  Mathews:  Century  Magazine,  1902,  pp.  783-792;  Science,  1902,  xl,  p.  492. 

3  Insectivorous  Plants,  p.  57. 

4  Unpublished  observations. 


io8  Shiro  Tashiro 

This  fact  lead  to  a  search  for  evidence  of  the  chemical  nature  of 
irritability  and  in  a  number  of  papers  5  it  was  clearly  pointed  out  that 
the  anaesthetics  were  probably  acting  directly  in  a  chemical  manner 
instead  of  indirectly,  by  affecting  permeability,  and  that  probably 
the  anaesthetics  acted  by  uniting  with  the  protoplasm  where  O2  usually 
took  hold.  This  view  was  strengthened  by  the  temperature  coeffi- 
cient of  conduction,  which  is  nearly  that  of  a  chemical  reaction;  by 
the  importance  of  O2  for  artificial  parthenogenesis;  and  by  many  other 
facts  some  of  which  have  recently  been  collected  by  Haberlandt, 
Buijtendijk  and  others. 

Although  it  has  been  established  by  repeated  demonstrations,  that 
the  nerve  does  not  fatigue  under  ordinary  conditions,  as  measured 
by  the  method  used  in  muscular  studies,  yet  Frohlich  6  observed  that 
the  nerve  undergoes  certain  changes  by  long  activity.  Gotch  and 
Burch  discovered 7  in  1889  that  if  two  stimuli  are  successively  set 
up  within  ^^  of  a  second,  only  one  negative  variation  is  produced. 
This  critical  interval,  or  refractory  period,  is  found  to  be  altered  by 
temperature  changes,  by  drugs,  asphyxiation,  and  anaesthetics.8 
Thus  by  prolonging  the  refractory  period  by  partial  anaesthesia,  Froh- 
lich easily  demonstrated  that  with  a  frequency  of  stimulation  less 
than  this  normal  refractory  period,  stimulation  of  the  attached  muscle 
no  longer  occurred.  He  interprets  this  as  a  phenomenon  of  fatiga- 
bility  of  the  nerve.  Thoner's  9  observation  seems  to  lead  to  a  similar 
interpretation,  for  he  found  recently  that  fatigability  is  less  effec- 
tive when  the  refractory  period  is  shortened  by  high  temperature. 
There  seems,  then,  to  be  fatigue  in  the  nerve,,  but  it  cannot  be 
measured  by  an  ordinary  scale. 

After  the  complete  failure  of  the  chemical  detection  of  CO2  and 

1  A.  P.  MATHEWS:  Biological  bulletin,  1904-5,  viii,  p.  333;  this  Journal, 
1904,  xl,  p.  455;  ibid.,  1905,  xiv,  p.  203;  Biological  Studies  by  the  pupils  of 
William  Sedgwick,  1906,  p.  81;  Journal  of  pharmacology  and  experimental 
therapeutics,  1911,  ii,  p.  234. 

6  FROHLICH:  Zeitschrift  fur  allgemeine  Physiologic,  1903-4,  iii,  p.  445.    Ibid., 

P-  75- 

7  GOTCH  and  BURCH:  Journal  of  physiology,  1899,  xxiv,  p.  410. 

8  See  TAIT  and  GUNN,  Quarterly  journal  of  experimental  physiology,  1908, 
i,  p.  191;  TAIT,  ibid.,  1909,  ii,  p.  157. 

9  THONER:    Zeitschrift  fur  allgemeine  Physiologic,  1908,  viii,  p.  530;    ibid*, 
1912,  xiii,  pp.  247,  267,  530. 


Carbon  Dioxide  From  Nerve  Fibres  109 

acids  in  the'  excited  nerve,  Waller  still  believes  that  it  must  give  off 
CO2  when  stimulated.  In  1896,  he  showed,  with  an  electro-physio- 
logical method,  that  among  other  reagents,  CO2,  in  minute  quanti- 
ties, increased  the  excitability  of  the  isolated  nerve  of  the  frog,  and 
that  the  normal  nerve,  when  excited,  also  increased  its  activity.10 
From  this  he  ingeniously  formed  the  hypothesis  that  every  activity 
in  the  nerve  fibre  must  be  associated  with  CO2  production. 

That  there  may  be  CO2  production  in  the  nerve,  but  too  small  to 
be  measured  by  ordinary  methods,  is  shown  by  the  following  calcu- 
lations: *A  frog  (Rana  temporaria)  gives  off  0.355  gram  of  CO2 
per  kilogram  per  hour  at.  19  —  20°  C.11  A  small  piece  of  the  nerve 
fibre  of  the  same  animal,  say  i  cm.  in  length,  will  weigh  in  the  neigh- 
borhood of  10  milligrams.  Now,  if  the  mass  of  the  nerve  respires  at  the 
rate  of  the  whole  animal,  it  would  give  off  about  0.0000007  grams  of 
CO2  during  ten  minutes.  This  calculation  at  once  suggested  that 
the  lack  of  positive  evidence  of  metabolism  in  the  nerve  fibre  was 
not  at  all  conclusive  that  such  metabolism  did  not  occur,  in  view  of 
the  limitation  of  the  methods  for  the  estimation  of  C02.  It  was 
evidently  necessary  to  devise  methods  for  the  detection  of  very 
minute  quantities  of  C02.  Thus  at  Professor  Mathews'  suggestion 
a  new  method  for  C02  analysis  was  first  devised,  and  then,  under 
his  direction,  I  have  undertaken  to  go  back  once  more  to  the  question 
of  CO2  production  in  the  nerve  fibre  during  the  passage  of  a  nerve 
impulse. 

To  study  the  nature  of  metabolism  involved  in  a  tissue,  one 
should  at  least  determine  the  oxygen  consumption  and  the  carbon 
dioxide  production.  Inasmuch,  as  the  present  problem,  however,  is 
concerned  only  with  direct  evidence  for  the  existence  of  metabolism 
in  the  nerve  fibre,  I  have  attempted  to  measure  C02  production 
only,  for  it  is  true  that  the  lack  of  oxygen  consumption  may  not 
necessarily  indicate  the  absence  of  chemical  changes,  while  the  pro- 
duction of  CO2  will  surely  prove  the  presence  of  metabolism.  Further- 
more, as  CO2  production  is  the  only  sure  universal  expression  of  the 
respiratory  activity  in  anaerobic  and  aerobic  plant  and  animal  tissue 
in  normal  condition,  the  inquiry  of  CO2  production  in  an  excited  nerve 
will  not  only  concern  the  problem  of  the  nature  of  the  nerve  impulse 

10  WALLER:  Croonian  lecture,  Philosophical  transactions,  London,  1896. 

11  Taken  from  Pott's  figures.     See  figures  in  Table  ix,  p.  129. 


no  Shiro  Tashiro 

itself,  but  may,  also,  aid  in  forming  a  fundamental  conception  of  the 
tissue  respiratory  mechanism.  In  this  way,  if  the  protoplasmic  irri- 
tability has  a  direct  connection  with  the  cellular  respiration,  then 
our  idea  of  the  general  nature  of  the  pharmodynamics  of  many 
reagents  on  a  living  tissue  may  be  essentially  modified. 


METHODS  AND  MATERIALS 

Two  new  apparati  were  constructed  which  will  detect  CO2  in  as 
small  quantities  as  one  ten-millionth  of  a  gram  and  estimate  it  with 
quantitative  accuracy.  The  detailed  method  has  been  described  in 
a  separate  article.12 

Preliminary  experiments  with  these  new  apparati  showed  that  the 
sciatic  nerves  of  dogs  gave  too  large  quantities  of  C02  for  my  method 
so  that  I  was  compelled  to  use  a  smaller  nerve  of  a  cold-blooded 
animal  for  quantitative  estimation.  For  exact  measurements  of  CO2 
production,  I  have  used  only  two  kinds  of  nerve,  although  I  have  used 
a  large  variety  of  nerves  in  qualitative  experiments.  For  a  non- 
medullated  nerve  fibre,  Prof.  G.  H.  Parker 13  was  so  kind  as  to  sug- 
gest to  me  that  I  use  the  nerve  trunk  of  the  claws  of  the  spider  crab 
(Labinia  Caniliculata)  which  is  a  bundle  of  mixed  sensory  and  motor 
fibres.  The  frog,  whose  sciatic  was  used  as  a  representative  for 
medullated  nerve,  was  exclusively  Rana  pipiens,  obtained  from 
Indiana. 

As  my  apparati  in  the  present  form  cannot  be  used  for  a  muscle 
nerve  preparation  nor  for  the  normal  nerve  in  situ,  the  use  of  an 
isolated  nerve  could  not  be  avoided.  Experimental  factors  thus  intro- 
duced should  be  carefully  considered  before  we  interpret  the  observa- 
tion as  a  normal  metabolism.  This  serious  objection,  however,  can  be 
overlooked,  as  far  as  our  fundamental  question  of  different  metabolic 
activities  before  and  after  a  stimulation  is  concerned,  for  Waller  14 
has  demonstrated  that  the  presence  of  excitability  in  an  isolated 
nerve  persists  as  long  as  nineteen  hours  provided  that  the  electrical 
changes  correctly  represent  the  state  of  excitability.  Although 

12  See  pp.  137-145- 

13  For  this  and  other  suggestions,  I  am  under  great  obligation  to  Dr.  Parker. 

14  WALLER:  1896,  Brain,  xix,  p.  53. 


Carbon  Dioxide  From  Nerve  Fibres  in 

Herzen  claims  that  under  cerlain  conditions  of  local  narcosis  the 
nerve  fibre  may  give  an  action  current  without  any  muscular  con- 
traction (Wedenshi  and  Boruttau  both  deny  this),  and  Ellinson  15 
recently  demonstrated  by  the  use  of  cinchonamine  hydrochloride 
the  absence  of  negative  variations  without  abolishing  the  excitability 
of  the  nerve,  yet  evidences  are  now  abundant  to  indicate  that  the 
action  current  is  a  normal  physiological  phenomenon  in  uninjured 
tissue  expressing  the  simultaneous  activity  resulting  in  a  corre- 
sponding change  in  the  peripheral  organ.16  These  facts,  therefore, 
must  be  taken  as  showing  that  as  long  as  a  negative  variation  remains, 
the  nerve  is  probably  excitable;  and  that  the  phenomena  observed  in 
the  isolated  nerve  could  be  regarded  as  identical  with  that  of  a  nor- 
mal nerve  as  far  as  the  passage  of  a  nerve  impulse  in  an  isolated 
nerve  fibre  is  concerned. 


CO-2  PRODUCTION  FROM  RESTING  NERVE 

In  this  study  of  the  metabolism  of  the  resting  nerve,  particular 
care  was  taken  to  select  those  fibres  which  were  free  from  nerve  cells. 
The  work  of  several  investigators 17  seems  to  indicate  that  tissue 
oxidation  is  primarily  concerned  with  the  cell  nucleus.  Inasmuch 
as  the  respiration  in  the  central  nervous  system  is  certain  18  and  the 
blood  supply  to  fibres  is  seemingly  scanty,  the  notion  persists  among 
certain  biologists  that  a  nerve  fibre  should  not  respire  since  it  has 
no  nucleus.  In  order  to  test  the  correctness  of  such  an  idea,  I  have 
studied  quantitatively  the  output  of  CO2  from  various  lengths  of  nerve 
which  are  known  to  be  free  from  nerve  cells.19  Here  is  the  result: 

15  ELLINSON:  Journal  of  physiology,  1911,  xlii,  p.  i. 

16  For  further  details,  see:   GOTCH  and  HORSLEY:   Philosophical  transactions 
of  the  Royal  Society,  1891,  clxxii,  p.  514;  BERNSTEIN:    Archiv  fur  die  gesammte 
Physiologic,  1898,  Ixxiii,  p.  376;   REID  and  MCDONALD:  Journal  of  physiology, 
1898-9,  xxiii,  p.  100 ;    LEWANDOWSKY:    Archiv  fiir  die  gesammte  Physiologic, 
1898,  Ixxiii,  p.  288;  ALCOCK  and  SEEMANN,  ibid.,  1905,  cviii,  p.  426. 

17  See  SPITZER:    Archiv  fiir  die  gesammte  Physiologic,  1897,  Ixvii,  p.  615; 
M.  NUSSBAUN:  Archiv  fur  mikroskopische  Anatomic,  1886,  xxvi,  p.  485;   R.  S. 
LILLIE:  This  Journal,  1902,  vii,  p.  412. 

18  L.  HILL:  Quoted  from  Hulliburton's  Chemistry  of  nerve  and  muscle,  p.  79. 

19  In  this  connection,  I  wish  to  express  my  indebtedness  to  Prof.  H.  H.  Donald- 
son for  his  kind  advice. 


ii2  Shiro  Tashiro 

Non-Medullated  Nerve  Fibre.  —  {The  nerve  of  the  spider  crab, 
and  apparatus  2  for  the  qualitative,  and  apparatus  i,  for  the  quanti- 
tative, estimations  were  used.)  When  I  place  the  nerve  of  a  spider 
crab  in  the  right  chamber  and  no  nerve  in  the  left,  and  watch  for  the 
deposit  of  barium  carbonate,  the  drop  on  the  right  will  soon  be  coated 
with  the  white  precipitate,  but  no  precipitate  whatever  is  visible  with 
a  lens  in  the  left.  CO2  is  thus  shown  to  be  produced  by  this  resting 
nerve.  Now,  by  interchanging  the  nerve  from  the  right  to  the  left, 
no  nerve  being  in  the  right,  we  can  convince  ourselves  of  the.  correct- 
ness of  this  conclusion,  by  eliminating  any  technical  error  which  might 
produce  the  different  results  in  different  chambers.  The  rate  at  which 
the  precipitate  appears  and  the  quantity  of  the  precipitate,  depends 
on  the  size  of  the  nerve.  In  fact,  CO2  production  from  the  resting 
nerve  of  the  spider  crab  is  found  to  be  proportional  to  its  weight, 
other  things  being  equal,  and  is  constant:  For  10  milligrams  per  ten 
minutes  it  gives  6.7  X  io-7  grams  at  15  —  i6°c. 

The  quantitative  determination  of  this  amount  is  made  in  the 
following  manner : 

The  claws  of  the  crab  are  carefully  removed,  and,  by  gejitly 
cracking  them,  the  long  fibre  of  the  nerve  trunk  is  easily  isolated. 
After  removing  the  last  drops  of  the  water  by  a  filter  paper,  the  nerve, 
with  the  aid  of  glass  chop  sticks,  is  carefully  placed  on  the  glass  plate,20 
and  quickly  weighed.  The  glass  plate  with  the  nerve  is  now  hung 
on  the  platinum  hooks  in  the  respiratory  chamber  A,  and  then  the 
chamber  sealed  with  mercury.  The  analytic  chamber  is  now  filled 
with  mercury  in  the  manner  described  elsewhere,21  and  then  the 
apparatus  is  washed  by  C02  free  air  as  usual.  The  time  when  the 
barium  hydroxide  is  introduced  to  the  cup  in  chamber  B  is  recorded, 
and  the  stop-cock  between  the  two  chambers  is  closed.  When  at 
the  end  of  ten  minutes  the  drop  at  cut  F  is  perfectly  clear,  having  not 
a  single  granule  of  the  precipitate  visible  to  a  lens,  thus  insuring  that 
the  air  is  absolutely  free  from  C02  then  a  known  portion  of  the  gas 
from  the  respiratory  chamber  is  introduced  into  the  chamber  below 
in  which  the  clear  drop  of  barium  hydroxide  has  been  exposed,  and-  it 
is  determined  whether  or  not  the  amount  of  the  gas  taken  contains 

20  The  weight  of  this  plate  is  known  so  that  the  weight  of  the  nerve  can  be 
determined  very  quickly.    See  p.  120. 

21  See  pp.  139. 


Carbon  Dioxide  From  Nerve  Fibres  113 

enough  CO2  to  give  the  precipitate  in  ten  minutes.  If  it  does,  a  fresh 
nerve  is  prepared  and  a  less  volume  of  the  gas  is  withdrawn;  if  it 
does  not,  a  larger  volume  should  be  taken  till  the  precipitate  appears 
within  ten  minutes.  (See  footnote,  page  140.) 

In  this  way,  by  repeated  experiments  with  several  fresh  nerves, 
a  minimum  volume  of  the  gas  for  a  known  weight  of  the  nerve  which 
gives  a  precipitate  is  determined.  This  minimum  volume  should 
contain  exactly  a  definite  quantity  of  C02  — namely  i.o  X  io-7 
gram.22 

In  this  way,  since  we  know  the  original  volume  of  the  respiratory 
chamber  from  which  this  minimum  volume  is  withdrawn,  and  since 
we  know  the  quantity  of  CO2  contained  in  this  volume,  it  is  easily 
calculated,  how  much  C02  is  produced  by  the  nerve  during  the  known 
period.  It  should  be  understood  that  in  determining  the  minimum 
volume  of  gas  taken  from  the  respiratory  chamber,  a  series  of  experi- 
ments were  conducted  in  order  to  calculate  both  the  minimum  volume 
which  just  gives  the  precipitate  and  the  maximum  volume  which 
does  not  give  the  the  precipitate  for  a  known  weight  of  the  nerve  for 
a  known  period  of  respiration  In  the  tables  following,  columns  8 
and  9  refer  to  these  volumes  calculated  from  experiments. 

Table  I,  gives  the  result  for  a  non-medulla  ted  nerve. 

Medullated  Nerve  Fibre.  —  For  the  quantitative  estimation  of 
C02  production  from  the  medullated  nerve  I  have  taken  a  frog's 
sciatic,  using  apparatus  2.  The  results  given  in  Table  II,  obtained  by 
similiar  methods,  show  that  each  ten  milligrams  of  the  frog's  sciatic 
nerve  gives  off  5.5  X  io~7  grams  for  the  first  ten  minutes. 

A  large  quantity  of  nerves  were  tested  and  it  was  determined 
whether  or  not  all  resting  nerves  give  off  CO2.  As  a  result,  I  found 
no  exception  in  any  of  them.  The  following  varieties  of  nerves 
were  examined: 

1.  MOTOR  NERVE:  Occulo-motor  nerve  of  the  skate.     (Raia  Ocallata.} 

2.  SENSORY  NERVE:  Olfactory  nerve  of  the  same.     (Raia  Ocallata.) 

3.  MEDULLATED  NERVE:    Sciatic  nerve  of  the  dog,  frog,  turtle,  mouse; 

optic  nerve  of  the  skate.     (Both  Raia  Ocallata  and  Raia  Erinecia.) 

4.  NON-MEDULLATED  NERVES:  Nerves  of  the  spider  crab;  olfactory  nerve 

of  the  skate.     (Raia  Ocallata.) 

5.  NERVE  OF  INVERTEBRATE:  Spider  crab's  nerves. 

22  See  p.  140. 


114 


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6.  NERVE  OF  VERTEBRATE  :  Nerves  of  frog,  dog,  mouse,  squiteague  (cynoscion 

Regalis),  and  skate.     (Both  Raia  Ocallata  and  Raia  Erinecia.) 

7.  NERVE  OF  WARM-BLOODED  ANIMALS:  Those  of  dog,  mouse  and  rabbits. 

8.  NERVE  OF  COLD-BLOODED  ANIMALS:  Frog,  squiteague  (cynoscion  Regalis) 

and  skate.     (Both  Raia  Ocallata  and  Raia  Erinecia.) 

From  this  I  have  concluded  that  isolated  nerves  of  all  animals 
give  off  CO2.  It  remains,  now,  to  consider  whether  this  C02  is  the 
product  of  normal  respiratory  activity  or  due  to  disintegration  of  the. 
dead  tissue. 


IS  THE   CO2   GIVEN  OFF  PRODUCED   BY  LIVING  PROCESSES? 

Comparison  of  Dead  and  Living  Nerves.  —  In  the  first  place,  it 
was  thought  that  if  C02  was  due  to  normal  metabolism  of  a  living 
nerve,  its  production  should  be  diminished  when  the  nerve  was  killed. 
The  following  result  (Table  III)  is  self  explanatory. 

TABLE  III 

COMPARISON  BETWEEN  NORMAL  AND  KILLED  (BY  STEAM)  NERVES  OF  SPIDER  CRAB 
12345  67 


Date 

Tempera- 
ture of 
room 

Weight  of 
nerve  in  mg. 

Stimula- 
tion 

c.c.  of  gas 
taken  from 
respiratory 
chamber 

Duration 
of 
respiration: 
minutes 

Ppt.  of 
Ba(C03) 
after  ten 
minutes 

Nov.  4 

13° 

40  (killed) 

no 

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10 

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40  (killed) 

st'n 

.5 

10 

- 

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16  (normal) 

no 

1. 

10 

+ 

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15 

16  (killed) 

no 

1. 

12 

- 

«     7 

16 

16  (normal) 

no 

1. 

10 

+ 

Comparison  of  Anaesthetized  and  Non-Anaesthetized  Nerves.  - 
It  is  naturally  feared,  however,  that  the  killing  experiment  itself  may 
not  prove  that  C02  production  is  necessarily  due  to  the  living  mechan- 
ism, for  high  temperature  may  .drive  off  CO2  produced  already  by  the 
process  of  tissue  disintegration,  just  as  the  CC>2  diffused  out  from  a 
wet  thread  saturated  with  the  gas,  the  rate  of  diffusion  being  a  func- 
tion of  temperature.  Thus  anaesthesia  was  tried,  although  we  should 


Carbon  Dioxide  From  Nerve  Fibres 


117 


expect  -at  the  outset  that  if  ether  had  no  direct  affect  on  the  respira- 
tory process,  as  some  physiologists  believe,  then  the  negative  results 
would  not  at  all  interfere  with  my  contention.  The  fact  is,  however, 
that  either  an  isolated  nerve  directly  treated  with  ether  vapor  or 
urathane,  or  the  nerve  isolated  from  a  deeply  anaesthetized  frog  gave 
a  much  less  quantity  of  CO2  than  the  normal  nerve  isolated  from  a 
normal  frog  whose  heart  has  been  cut  away  for  a  period  of  time  equal 
to  that  of  etherization.  Anaesthetics,  then,  diminish  CO2  production 
from  an  isolated  nerve  fibre.  These  experiments  are  being  continued 
quantitatively. 

CO 2  Production  of  Isolated  Nerve  at  Successive  Time  Intervals. 

-  It  was  also  thought  that  if  CO2  production  was  due  to  bacterial 

decomposition,  although  it  is  highly  improbable  for  such  a  fresh 

tissue,  we  may  expect  that  either  killing  by  steam  or  treating  with 

TABLE  IV 

SHOWING  DECREASED  CO2  PRODUCTION  BY  LONG-STANDING  (FROG'S  SCIATIC) 
123  4 


Temperature 
of  room 

Time  elapsed 
after  isolation 

Minimum  c.c. 
necessary  to  give 
\,  calculated  for 
10  mgs.  10  minutes 

Total  CO2  pro- 
duced from  nerve 
of  10  mg.  for  10 
minutes 

24° 

immediately 

2.7  c.c. 

5.5  X  10-7g.  CO2 

25 

1  hour 

7.08  c.c. 

2.1  X  10-7g.  C02 

24 

2  hours 

10.8  c.c. 

1.4  X  10-7g.  CO2 

24 

5.5  hours 

12.8  c.c. 

1.1  X  KHg.  C02 

23.5 

7     hours 

15.3  c.c. 

.9  X  107g.  CO2 

23.5 

10.5  hours 

21.0  c.c. 

.6  X  10-7g.  C02 

24 

26     hours 

9.    c.c.1 

1.6  X  10-7g.  CO2 

24 

27.4  hours 

1.8.  c.c 

8.1  X  10-7g.  C02 

1  The  gradual  increase  at  this  point  should  be  noted  (after  26  hours,  it  is  clear  that 
bacterial  decomposition  sets  in). 

ether  would  check  the  C02  production,  and  that  the  results  observed 
above  may  not  necessarily  prove  that  C02  production  from  the  isolated 
nerve  fibre  is  due  to  a  respiratory  process.  Hence  a  number  of  the 
nerves  were  isolated  from  several  frogs  of  the  same  size  and  sex,  and 


n8  Shiro  Tashiro 

were  left  in  Ringer's  solution,  and  then  the  rate  of  the  gas  production 
is  determined  with  the  different  nerves  removed  at  successive  inter- 
vals of  time  from  the  Ringer's  solution  for  twenty-five  hours.  The 
interesting  results  given  in  Table  IV  not  only  show  that  CO2  from  the 
fresh  nerve  is  not  due  to  bacterial  decomposition,  but  it  also  indicates 
that  when  such  abnormal  decomposition  sets  in,  the  output  of  gas 
takes  a  sudden  jump.  This  Table  further  shows  that  the  vital  process 
by  which  CO2  is  produced  gradually  slows  up  as  the  tissue  approaches 
death,  indicating  that  the  decrease  of  CO2  production  is  parallel  to 
the  decrease  of  irritability  of  the  nerve. 

Increase  of  CO2  on  Stimulation.  —  -  The  most  convincing  evi- 
dence of  all  that  CO2  is  formed  by  a  vital  process  is  the  fact  that 
a  stimulated  nerve  gives  off  more  CO2  (Part  II)  indicating  the 
presence  of  normal  metabolism  in  the  living  nerve  which  is  accelerated 
when  the  nerve  is  stimulated.  Thus  we  may  safely  conclude  here 
that  like  any  other  tissue  or  organs,  the  nerve,  too,  respires  whether 
it  has  a  nucleus  or  not,  and  that  the  rate  of  C02  production  is  pro- 
portionate to  its  weight,  other  things  being  equal. 


C02  PRODUCTION  FROM  STIMULATED  NERVE 

We  have  now  come  to  our  main  inquiry,  namely,  is  there  any 
chemical  basis  for  irritability?  Just  what  relation  exists  between 
nervous  activity  and  chemical  changes  is  the  question  that  a  biologist 
should  consider  before  he  attempts  to  build  any  conception  of  the 
real  dynamics  of  living  matter.  For  it  is  the  phenomena  of  excita- 
bility in  the  nerve  fibre  that  has  stood  so  long  in  the  path  of  under- 
standing protoplasmic  irritability  in  general.  As  for  the  brain,  it  is 
now  established  that  certain  chemical  changes  are  involved  during 
stimulation  and  that  definite  chemical  changes  are  associated  with 
pathological  cases  either  in  its  chemical  composition  23  or  in  the  for- 
mation of  abnormal  metabolites.24  Aside  from  the  confused  facts 
concerning  histological  changes  in  the  ganglion  cells  of  fatigued  ani- 
mals, Hill  has  observed,  using  Ehrlich's  method  of  methylene  blue 

23  KOCH  and  MANN:  Archiv  of  neurology  and  psychiatry,  1909,  iv,  p.  44. 

24  DIXSON:  Journal  of  physiology,  1899-1900,  xxv,  p.  63;  CROFTAN:  American 
journal  of  the  medical  sciences,  1902,  p.  150. 


Carbon  Dioxide  From  Nerve  Fibres  119 

for  the  determination  of  the  rate  of  oxidation,  that  a  spot  of  cerebral 
surface,  if  stimulated,  loses  its  blue  color  owing  to  the  using  up  of  the 
oxygen.2'  In  case  of  the  nerve  fibre,  however,  we  have  already  seen 
that  no  direct  evidence  has  ever  been  presented  to  show  any  chemical 
changes  connected  with  its  activity,  although  there  has  been  some 
indirect  evidence.  As  considered  before,  the  failure  of  the  direct 
detection  of  CO2  from  the  stimulated  nerve  must  be  due  to  the  lack 
of  a  delicate  method.  Thus  using  the  new  method  we  have  already 
demonstrated  that  a  resting  nerve  gives  off  C02,  and  will  now  attempt 
to  prove  that  nerves  give  off  more  CO2  when  stimulated.26 

Electrical  Stimulation  of  non-Medullated  Nerve.  —  Owing  to  the 
scope  of  delicacy  of  the  new  method,  which  is  sensitive  to  as  small  a 
quantity  as  i.o  X  icr7  gram  (an  amount  corresponding  to  the  CO2 
contained  in  -g-  cc.  of  pure  air),  the  utmost  caution  must  be  taken  to 
prevent  any  complication  which  may  result  in  formation  or  absorption 
of  minute  quantities  of  C02.  After  I  had  found  by  experiment  that 
there  is  no  appreciable  increase  of  C02  due  to  the  direct  electrical 
decomposition  in  the  nerve  when  stimulated  by  a  weak  induction 
current  and  that  several  other  forms  of  stimulation  qualitatively 
confirmed  the  results  obtained  by  the  electrical  stimulation,  I  have 
naturally  employed  the  induction  current  as  a  stimulant  in  all  my 
experiments  on  the  quantitative  estimation  of  C02  production  from 
the  stimulated  nerve.27 

As  Table  V  shows,  the  stimulated  non-medullated  nerve  fibre  of 
the  spider  crab  gives  off  16.  X  io~7  grams  of  C02  for  10  milligrams  of 

25  HILL:  loc.  cit. 

26  Professor  Carlson  has  very  kindly  called  my  attention  to  a  recent  publica- 
tion from  the  Physiologisch  Laboratorium  der  Utrechtsche  Hoogeschool,  in  which 
Buijtendijk  reports  that  certain  head  nerves  of  fishes  take  up  more  O2  when 
electrically  stimulated.     He  could  not,  however,  find  any  increase  of  O2  con- 
sumption  in   the   sciatic   of   the   frog.     Also   see:    Koninklijk  Akademie   van 
Wetenschappen,  Amsterdam,  afd,  xix,  pp.  615-621. 

Haberlandt  also  recently  reports  (Archiv  fur  Physiologic;  1911,  p.  419)  that 
the  resting  nerve  takes  up  of  02,  41.7  —  33.4  cmm.  at  19°  —  24°  per  gram  per 
hour.  When  this  nerve  is  excited,  intake  of  O2  is  increased.  Since  the  respira- 
tory quotient  of  the  stimulated  nerve  is  equal  to  that  of  the  resting,  he  con- 
cludes that  when  the  nerve  is  excited,  it  must  give  off  more  COa.  He  does 
not,  however,  indicate  how  much  CO2  is  produced  by  stimulation. 

27  Use  of  non-polarizable  electrodes  was  impossible  for  my  apparatus,  for  the 
presence  of  foreign  liquid  in  the  chamber  interferes  with  C02  estimation.     As 


i2o  Shiro  Tashiro 

x- 

nerve  for  ten  minutes,  while  a  fresh  resting  nerve  gave  only  6.7  by 
io~7  grams  for  the  same  units.  The  details  of  the  methods  are  as 
follows : 

The  nerve  of  the  claw  of  the  spider  crab  is  isolated  as  before.  A 
comparative  estimation  was  made  first.  Two  pieces  of  the  nerve  of 
equal  weights  and  length  were  placed  separately  on  the  two  glass 
plates,  each  nerve  being  laid  across  the  electrodes  of  the  plate,  in  the 
manner  shown  in  Figure  i .  In  this  way  either  nerve  can  be  stimulated 
at  will.  These  glass  plates  are  hung  by  their  wires  upon  the  platinum 
wires  fused  into  the  side  of  the  apparatus,  these  wires  being  con- 
nected in  turn  with  the  induction  coil.  Under  this  condition,  when 
both  nerves  are  not  stimulated,  the  amounts  of  the  precipitate  .are 
equal  in  both  chambers.  However,  when  one  of  the  nerves  is  elec- 

0 


FIGURE.  1.  Glass  weighing  plate.  A.  B.  Platinum  wire  fused  in  the  rear  of 
the  glass  plate,  with  hooks.  C.  The  nerve  which  is  stimulated  at  D. 
G.  The  plate  proper. 

I  have  the  other  piece  of  the  same  glass  out  of  which  this  plate  is  made.  This 
piece  of  glass  is  weighed  exactly  equal  to  this  weighing  plate,  so  that  any 
wet  tissue  can  be  weighed  very  quickly.  In  order  to  make  results  more  accu- 
rate, no  attempt  was  made  to  weigh  closer  than  \  milligram. 

trically  stimulated  (the  distance  between  the  primary  and  secondary 
coils  was  always  more  than  10  cm.  using  a  red  dry  battery,  the  current 
being  barely  perceptible  on  the  tongue),  not  only  does  the  precipitate 
appear  sooner  in  the  chamber  in  which  the  excited  nerve  is  placed, 
but  also  the  quantity  of  the  carbonate  is  much  greater. 

To  test  whether  the  increase  of  COa  production  from  the  stimu- 
lated nerve  is  due  to  the  direct  decomposing  influence  of  the  current, 
or  to  the  increase  of  metabolism  produced  by  the  passage  of  a  nerve 

long  as  we  are  not  concerned  with  the  electrical  changes  in  the  nerve,  the  use  of 
platinum  electrodes  instead,  is  not  a  great  objection,  provided  that  the  current 
is  weak  enough  not  to  decompose  the  tissue  directly,  and  that  the  duration  of 
stimulation  is  not  very  long. 


Carbon  Dioxide  From  Nerve  Fibres  121 

impulse,  the  following  experiments  were  performed.  If  we  assume 
that  the  condition  under  which  an  electrical  decomposition  takes 
place  is"  the  same  both  in  the  living  and  the  dead  nerve,  then  if  the 
increased  CO2  is  due  to  the  current  itself,  we  should  expect  that  when 
a  killed  nerve  is  stimulated  by  a  current,  it  ought  to  increase  C02 
production  just  as  much.  When  I  placed  two  nerves  killed  by  steam 
in  each  chamber,  and  stimulated  only  one  of  them,  the  stimulated 
nerve  did  not  give  any  more  CO2  than  the  unstimulated,  using  the 
same  strength  of  current  employed  in  the  other  experiments.  In 
the  next  place,  it  was  thought  that  if  the  increase  of  CO2  is  due  to 
direct  electrical  decomposition,  not  limited  to  the  point  of  contact 
with  the  electrodes,  we  ought  to  get  a  proportional  increase  of  CO2 
by  altering  the  distances  through  which  the  current  directly  passes. 
The  fact  was,  however,  that  we  could  produce  an  increase  of  CO2 
production  by  stimulating  with  electrodes  2  mm.  apart  as  well  as  by 
15  mm.  apart.  Increase  of  CO2,  therefore,  is  due  to  nervous  excita- 
tion and  not  to  the  direct  influence  of  the  electric  current  itself. 

With  this  consideration,  I  have  proceeded  to  make  a  quantitative 
estimation  of  CO2  from  the  stimulated  nerve  in  the  manner  described 
before.  The  results  are  shown  in  Table  V. 

Electrical  Stimulation  of  Medullated  Nerve.  —  With  apparatus 
2,  the  output  of  CO2  from  the  excited  sciatic  nerve  of  the  frog  has  been 
quantitatively  estimated.  As  shown  below,  10  mgs.  of  the  sciatic 
nerve  gives  off  14.2  X  io~7  grams  of  CO2  during  ten  minutes  stimula- 
tion while  the  resting  nerve  of  the  same  animal  gave  off  5.5  X  io~7 
grams  for  the  same  units. 

Mechanical  Stimulation.  — •  We  have  now  established  the  fact  that 
when  a  nerve  is  stimulated  by  an  electrical  stimulus,  it  gives  off  more 
C02.  In  order  to  prove  more  conclusively  that  this  CO2  production 
is  due  to  the  passage  of  a  nerve  impulse,  I  have  employed  several 
other  means  which  are  known  to  have  definite  influence  on  excita- 
bility of  the  nerve.  So  far,  the  use  of  these  methods  has  been 
confined  to  qualitative  experiments,  but  the  results  are  a  sufficient 
confirmation  of  the  observations  made  by  electrical  stimulation.  I 
cite  them  here  as  a  preliminary  report. 

Since  the  ordinary  method  for  mechanical  stimulation  cannot  be 
applied  directly  to  the  nerve  in  my  apparatus  in  its  present  form,  I 
used  a  different  method,  namely,  crushing  the  nerve.  That,  when  a 


122 


Shiro  Tashiro 


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124  Shiro  Tashiro 

protoplasm  is  smashed,  there  occurs  vigorous  chemical  changes,  is 
shown  by  several  investigators.  Fletcher 28  reports  that  injured 
muscle  gives  off  more  CO2  than  the  normal. 

Later  he  and  Hopkins 29  discovered  that  muscle,  under  a  similar 
condition,  is  richer  in  lactic  acid. 

Dr.  Mathews  has  observed  a  similar  activity  in  crushed  eggs  of 
Arbacia.  Quite  accidently,  I  have  discovered  that  a  fresh  nerve,  too, 
when  crushed  with  the  rough  edge  of  a  glass  rod  gives  off  more  C02- 
This  increase  of  gas  production  from  the  injured  nerve,  I  take  to  be 
due  to  mechanical  stimulation.  To  test  this  hypothesis,  I  rendered 
the  nerve  unexcitable  by  means  of  ether  and  0.2  m.  solution  of  KC1, 
which  is  known  to  abolish  excitability  of  a  nerve.30 

Under  these  conditions,  I  gbserved  no  increase  of  gas  production 
when  the  nerve  is  crushed.  Therefore,  the  metabolism  existing  in 
the  living  nerve  must  be  accelerated  by  this  stimulation  when  it  is 
injured. 

This  interpretation,  however,  is  not  accordant  with  that  of  Fletcher 
and  Hopkins,  on  muscle.  In  studies  of  lactic  acid  formation  in  muscle, 
they  found  that  lactic  acid  is  spontaneously  developed,  under  anae- 
robic condition,  in  excised  muscle,  and  that  fatigue  due  to  contractions 
of  excised  muscle  is  accompanied  by  an  increase  of  lactic  acid.  In 
an  atmosphere  of  62,  there  is  no  survival  development  of  lactic  acid 
for  long  periods  after  excision.  From  a  fatigued  muscle,  placed  in 
O2,  there  is  a  disappearance  of  lactic  acid  already  formed.  But  this 
disappearance  of  lactic  acid,  due  to  oxygen,  does  not  occur,  or  is 
masked,  at  supraphysiological  temperature  (e.  g.,  at  30  ).  Now 
traumatic  injury  to  an  irritable  muscle  too  produces  a  rapid  develop- 
ment of  acid.  Since,  however,  in  this  case  the  disappearance  of  lactic 
acid  due  to  O2  does  not  occur,  they  conclude  that  one  essential  condi- 
tion for  this  effect  of  oxygen  appears  to  be  the  maintenance  of  the 
normal  architecture  of  the  muscle.  Thus  they  contend  that  the 
increase  of  the  lactic  acid  by  mechanical  injury  is  not  due  to  stimula- 
tion, but  must  be  due  to  tissue  destruction. 

They,  however,  did  not  determine,  as  far  as  I  know,  how  much 
the  output  of  CO2  is  affected  by  treating  the  injured  tissue  with  O2. 

28  FLETCHER:  Journal  of  physiology,  1898-9,  xxiii,  p.  37. 

29  FLETCHER  and  HOPKINS:  ibid.,  1906-7,  xxxv,  pp.  261,  288. 

30  MATHEWS:  This  Journal,  1904,  xi,  p.  463. 


Carbon  Dioxide  From  Nerve  Fibres  125 

Unless  it  is  proven  that  CO2  production  from  the  injured  muscle  is 
quantitatively  equivalent  to  lactic  acid  formed,  their  interpretation 
cannot  be  applied  to  the  injured  nerve,  for  in  the  case  of  the  "plateau  " 
of  the  survival  muscle  respiration,  when  in  complete  loss  of  irritability, 
the  lactic  acid  yield  remains  stationary,  Hill  calculated  that  the  C02 
production  corresponds  to  the  amount  liberated  from  the  carbonate 
of  the  tissue  by  the  lactic  acid  formed.31 

Furthermore,  if  their  interpretation  is  applied  to  the  nerve,  the 
fact  that  etherized  nerves  or  nerves  rendered  unexcitable  by  KC1  do 
not  increase  CO2  output  when  crushed,  cannot  be  explained.  The 
fact  that  only  excitable  nerves  when  injured  increase  their  CO2  pro- 
duction, is  a  sufficient  proof  that  some  sort  of  stimulation  is  applied 
to  the  nerve  when  crushed,  the  tissue  destruction,  no  doubt,  following 
afterward.  The  increase  of  CO2  production  on  crushing  the  living 
nerve  and  its  absence  on  crushing  the  anaesthesized  nerve  is  the  point 
that  I  want  to  emphasize  here  in  order  to  confirm  my  results  obtained 
by  electrical  stimulation.  I  may  add  here  that  a  perfectly  parallel 
increase  of  CO2  by  crushing  has  been  observed  in  dry  seeds,  including 
wheat,  wild  oats,  Lincoln  oats,  Swedish  select  oats,  leaves  of  Japanese 
ivy,  and  spinal  cords  of  rabbit.32 

Chemical  Stimulation.  —  The  study  of  the  nature  of  chemical 
stimulation  has  been  so  thoroughly  made  33  that  at  first  it  was  thought 
that  chemical  reagents  would  be  ideal  as  stimuli. 

It  was  soon  discovered,  however,  that  the  presence  of  minute 
quantities  of  a  foreign  liquid  is  such  a  disturbing  factor  that  stimula- 
tion by  salt  solutions  could  not  be  used  for  quantitative  experiments. 
With  a  qualitative  analysis,  however,  I  found  a  variety  of  evidences 
which  show  that  the  nerve  stimulated  chemically  gives  off  more  CO2, 
and  that  the  nerve  rendered  less  excitable  by  reagents  decreases  C02 
production. 

When  each  sciatic  nerve  of  a  frog  is  isolated  and  one  is  left  in  the 
normal  saline  in  one  case,  and  in  the  body  of  the  frog  in  the  other,  for 
the  same  length  of  time,  and  then  transferred  to  the  two  chambers  of 
the  apparatus,  if  the  quantities  of  the  precipitate  are  compared,  it  is 
found  that  the  nerve  which  has  been  in  normal  saline  gives  more  CO2. 

31  HILL:  Journal  of  physiology,  1912,  xliv,  p.  481. 

32  Fuller  discussion  of  these  will  appear  in  a  subsequent  paper. 

33  MATHEWS:  This  Journal,  1904,  xl,  p.  455;  1905,  xiv,  p.  203. 


126  Shiro  Tashiro 

It  is  known  that  normal  saline  stimulates  frog's  sciatic  nerves.  The 
different  rates  at  which  CO2  is  produced  from  the  different  nerves 
treated  by  various  concentrations  of  KC1  is  equally  instructive.  It 
is  known  that  when  a  nerve  is  placed  in  a  molecular  solution  of  KC1, 
a  stimulation  takes  place  for  a  considerable  time.  Then  it  finally 
becomes  unexcitable,34  whereas,  .2  m.  KC1  solution  abolishes  nervous 
excitability  in  a  short  time  without  primary  stimulation.  The  CO2 
production  follows  exactly  analogous  to  this.  The  nerve  treated  with 
the  stronger  solution  gives  more  CO2  than  that  of  a  weaker  solution. 
This  was  true  even  after  both  nerves  became  unexcitable,  showing 
that  the  nerve  must  be  giving  off  more  CO2  while  being  stimulated 
by  the  stronger  solution.  Although  my  quantitative  data  are  not 
complete  at  this  stage,  this  preliminary  statement  is  sufficient  to  show 
that  the  nerve  chemically  stimulated  gives  off  more  CO2.  It  may 
be  added  in  passing  that  the  different  solubility  of  CO2  in  the  different 
concentrations  of  these  salts  solutions  cannot  explain  these  results 
solely  by  a  physical  interpretation,  for  there  is  not  enough  difference 
in  the  solubility  of  CO2  in  dilute  equimolecular  solutions  of  KC1, 
and  NaCl,  whose  effect  on  CO2  production  is  so  divergent,  the  former 
salt  diminishing,  the  latter  increasing  it. 

Heat  Stimulation.  —  It  may  be  recalled  in  Table  I  that  high  tem- 
perature increases  the  output  of  CO2  from  the  resting  nerve.  A 
respiratory  process  should  increase  proportionally  to  the  temperature. 
Raising  of  temperature,  however,  not  only  increases  the  rate  of  res- 
piration, but  also  (particularly  by  sudden  changes  of  it)  stimulates 
the  nerve.  A  very  interesting  fact  is  observed  in  connection  with 
the  killing  of  the  nerve.  When  the  nerve  is  killed  gradually  by  a 
slow  increase  of  temperature,  it  gives  off  more  C02  than  when  killed 
suddenly,  the  determination  being  made  after  both  are  killed.  CO2 
production  from  the  dead  nerve  under  this  condition  must  be  due  to 
the  diffusion  of  the  gas  which  was  formed  previously,  just  as  Fletcher's 
dead  muscle  is  charged  with  CO2  gas.  The  different  outputs  of  C02 
between  slowly  killed  and  suddenly  killed  nerves  cannot  be  accounted 
for  unless  we  assume  that  in  one  case,  C02  is  produced  more  while 
being  killed  than  in  the  other.  Whether  such  increase  of  C02  produc- 
tion, however,  was  due  to  the  acceleration  of  normal  respiration  by 
the  slowly  increasing  temperature,  or  due  to  direct  stimulation  caused 

34  MATHEWS:  loc.  cit. 


Carbon  Dioxide  From  Nerve  Fibres 


127 


by  heat,  or  due  to  both,  cannot  be  decided  here  unless  we  consider  the 
relation,  between  excitation  and  tissue  respiration.35 

It  is  hoped  that  we  may  have  a  better  understanding  of  this  matter 
when  we  study  the  temperature  coefficient  of  normal  respiration  of  the. 
nerve.  At  present,  we  are  satisfied  to  state  only  that  there  is  a  strong 
evidence  to  support  the  conclusion  that  heat,  too,  increases  CO2 
production  from  the  nerve. 


DISCUSSION   OF   THE   RESULTS 

Comparison  of  Metabolism  of  Non-Medullated  and  Medullated 
Nerve.  —  Although  it  appears  ridiculous  to  attach  any  significance  to 
the  marked  similarity  in  the  magnitudes  of  CO2  production  from  non- 
medullated  and  medullated  nerves,  the  temptation  is  irresistible  to 
comment  on  the  high  output  of  CC>2  from  the  non-medullated  nerve 
fibre.  Let  us  study  the  Table  following  (Table  VIII),  in  which  a 
summarized  comparison  is  given. 

TABLE  VIII 


Nerve 

CO2  from 
resting  nerve 

CO2  from 
stimulated  nerve 

Rate  of 
increase 
of  CO2 

Non-medullated 
(spider  crab) 
Medullated  (frog) 

6.7  X  1O7  g.  (15°  -  16°  ) 
5.5  X  10-7  g.  (19°  -  20°  ) 

16.  X  10-7  g.  (14°    -  16°) 
14.2  X  10-7  g.  (20°  -  22°) 

2.4  times 
2.6     " 

Since  I  have  found  that  injury  increases  the  C02  production  from 
the  nerve,  the  values  I  have  obtained  from  cut,  or  isolated,  fresh 
resting  nerves,  such  as  I  had  to  use,  may  be  somewhat  greater  than 
the  output  of  normal  uninjured  nerves  would  be.  But  since  Alcock36 
has  shown  that  a  non-medullated  nerve  gives  a  higher  electrical 
response,  both  in  the  negative  variation  and  the  injury  current,  the 
CO2  increase  due  to  the  cut  alone  will  probably  be  greater  in  case  of 
the  non-medullated  nerve  than  in  that  of  the  medullated  one.  That 
means  that  the  value  of  the  CC>2  production  for  the  resting  uninjured, 

35  See  p.  134. 

36  ALCOCK:  Proceedings  of  the  Royal  Society,  1904,  Ixxiii,  p.  166. 


128  Shiro  Tashiro 

non-medullated  nerve  should  be  reduced  more  from  the  figures  found 
for  the  isolated  nerve,  than  that  of  the  medullated  one.  In  other 
words,  by  lowering  6.7  X  io-7  gram  which  is  the  value  for  resting,  non- 
. medullated,  isolated  nerves,  the  rate  of  increase  of  CO2  by  stimula- 
tion in  the  uninjured  nerve  would  become  higher  than  2.4  times,  and 
probably  higher  than  2.6  times,  which  is  the  rate  for  the  medullated 
nerve.  This  greater  effect  in  the  non-medullated  nerve  is  what  we 
should  expect  if  our  present  conception  that  conduction  is  in  the 
axis  cylinder  only,  is  correct.  Before  any  accurate  comparison  of 
the  increase  of  C02  production  on  stimulation  of  non-medullated  and 
medullated  nerves  can  be  made  it  will  be  necessary,  however,  to 
determine  how  much  of  the  CO2  from  the  resting  nerve  is  due  to  injury 
alone.  Before  we  consider  this  point  seriously,  also,  we  should  deter- 
mine the  metabolic  activities  of  greater  numbers  of  nerves  of  different 
animals.  Such  an  investigation  is  at  present  useless  until  we  deter- 
mine more  quantitatively  the  relation  between  CO2  production  and 
the  various  strengths  of  stimulation  and  the  degree  of  excitability. 
If  any  uniformity  of  C02  output  in  respect  to  anatomical  varia- 
tions is  discovered,  light  may  be  thrown  on  the  function  of  the 
medullary  sheath  and  other  differentiations. 

However  insignificant  these  results  may  be  as  far  as  the  similar 
rates  of  the  gas  production  of  these  two  nerves  is  concerned,  it  should 
be  strongly  emphasized  that  technical  error  plays  no  part  in  these 
determinations.  Inasmuch,  as  we  are  dealing  with  such  an  extremely 
small  amount  of  the  gas,  it  is  quite  natural  for  those  who  are  not 
familiar  with  my  apparati  to  suspect,  by  a  hasty  inspection  of  my 
results,  that  the  small  differences  I  found  under  different  metabolic 
conditions  may  be  due  to  mere  experimental  variations.  For  this 
reason,  particular  attention  is  called  to  a  detailed  description  of  the 
quantitative  method  I  used,  especially  the  footnote  on  page  144, 
where  I  have  cited  a  series  of  determinations  of  unknown  quantities 
of  CO2  in  testing  my  apparati.  I  may  repeat  here  that  my  experi- 
ments with  the  spider  crab  and  the  winter  skate  were  done  at  Woods 
Hole37  during  the  summer  of  1911,  while  those  with  the  frog  were 
done  in  Chicago  during  the  winter  of  1912.  Under  these  different 
conditions,  I  have  not  only  used  the  different  sizes  of  nerves,  but  also 

37  I  take  great  pleasure  in  acknowledging  my  indebtedness  for  the  kind  accom- 
modation offered  me  by  Drs.  Lillie  and  Drew  at  Woods  Hole. 


Carbon  Dioxide  From  Nerve  Fibres 


129 


experimented  with  two  different  apparati,  the  respiratory  chambers 
of  which  have  had  entirely  different  capacities.38 

Comparison  between  the  Metabolism  of  Resting  Nerves  and  that 
of  Other  Tissues.  —  To  compare  the  rate  of  metabolism  of  the  nerve 
with  that  of  other  tissues  is  a  matter  of  no  great  physiological  value 
on  account  of  great  variations  which  do  not  affect  equally  the  rate 
of  CO2  production.  Simply  to  give  a  better  picture  of  the  scope  of 
nervous  metabolism,  however,  let  us  make  the  following  comparison: 
Since  there  is  no  exact  determinations  made  on  either  the  other  organs, 
or  the  whole  animal,  in  the  case  of  the  spider  crab,  I  have  quoted  those 
of  the  nearest  Crustacea  of  which  data  are  available.  (Table  IX). 

TABLE  IX 


Animals 

CO2  per  Kg. 

per  hour 

Temperature 

Determined  by  l 

Crustacea  (whole  animal) 
Cray  fish  (Astacus) 

37  7  c  c 

12°  5 

Jolyet  and  Regnaut 

((                  It                    It 

Crab  (Cancer  pagurus) 

899  cc 

16 

n           (i              te 

L,obster  (Homarus  vulgaris)  .... 

^erve  of  spider  crab  (Labinia  cani- 
liculata] 

54.4  c.c. 
212  cc 

15 
15°  -  16° 

(t           ti              (t 

Tashiro 

Frog: 
(Rana  esculenta)  (whole  animal)  . 

(Rana  temporaria)  (whole  animal) 
(Rana  pipiens)  (sciatic  nerve)     . 

(Rana       temporaria  2)     (isolated 
muscle)     

.082  gms. 
.355    " 
.  .33      " 

.18      " 

17 
19°  -  20° 
15 

21 

Schultz 
Pott 
Tashiro 

Fletcher 

Dog  . 

1.325    " 

Regnaut  and  Reiset 

Man  at  rest     

.41      " 

Pettenkoffer  and  Voit 

«      «     a 

61      " 

t(             (t      n 

«      «     a 

37      " 

Speck 

1  All  the  figures  are  quoted  from  Schafer's  Text  Book  of  Physiology  i,  pp.  702,  707  and 
708,  except  that  of  the  isolated  muscle  which  I  calculated  from  Fletcher  (loc.  cit.).  Fletcher 
fails  to  state  the  weight  of  a  leg,  but  gives  the  value  .2  c.c.  for  one-half  hour.     Hill  believes 
that  if  we  take  each  leg  6  g.  in  average,  the  value  will  not  be  far  from  the  truth. 

2  Fletcher  fails  to  state  the  species  of  the  frog,  but  it  is  inferred  from  Hill's  paper. 

38  See  the  last  columns  of  Table  I  and  Table  II. 


130  Shiro  Tashiro 

Active  Nerves.  —  That  the  nerve  increases  its  CO2  production 
approximately  2.5  times  when  stimulated,  is  in  accordance  with  our 
conception  of  the  metabolism  of  other  acting  organs.  Just  how  much 
increase  of  CO2  takes  place  during  functional  activity  of  an  organ  or 
organisms  depends  on  conditions  as  well  as  on  habits  of  different  organs 
and  animals.  Pettenkofer  and  Voit39  report  that  a  man  (weighing 
70  kgs.)  gives  off  when  working  0.76  grams  per  kg.  per  hour,  while 
resting  only  .56  gram.  Barcroft40  found  that  the  submaxillary  gland 
when  stimulated  by  the  chorda  tympani  gives  off  3-7  times  more 
.CO2  than  the  resting  gland.  In  the  case  of  contracting  muscle,  the 
results  are  very  contradictory.  Hermann  41  found  that  the  contract- 
ing muscle  gave  off  9.3  per  cent  of  CO2  (by  volume)  while  the  resting 
one,  only  1.4  per  cent.  Tissot 42  and  other  workers  also  found  a 
similar  increase  of  CO2  from  contracting  muscle.  Minot,43  working 
with  Ludwig,  maintains  that  there  is  no  relation  whatever  between 
CO2  production  and  muscle  tetanus.  L.  Hill 44  and  Fletcher 45  both  con- 
firmed Minot's  work  by  finding  no  increase  of  CO2  production  from 
muscular  tetanus.  According  to  Fletcher,  the  increase  he  found  in 
CO2  production  from  a  contracting  muscle  in  a  closed  vessel  is  due  to 
the  rigor.  Under  this  condition,  he  believes,  increased  formation  of 
lactic  acid  is  responsible  for  liberating  C02  already  produced.  In 
either  case,  it  is  understood  that  functional  activity  in  the  muscle  is 
accompanied  by  an  increase  of  metabolic  activity.  It  is  difficult  to 
compare  this  increase  of  metabolic  activity  of  the  muscle  with  that 
of  the  nerve  unless  we  determine  how  much  and  what !  ind  of  metabol- 
ism takes  place  in  contracting  muscle. 

Respiration  Quotient  of  the  Nerve  Fibre.  —  As  quoted  before 
Haberlandt  found  that  a  resting  nerve  consumes  41.7  to  83.4  cmm. 
02  for  i  gm.  for  an  hour  at  19°  -  24°.  Although  he  has  not  deter- 
mined chemically  the  production  of  CO2  he  could  easily  read  the 
respiration  quotient  by  means  of  the  index  fluid.  Thus  he  found 

39  PETTENKOFER  and  VOIT:  loc.  cit. 

40  BARCROFT:  Ergebnisse  der  Physiologic,  1908,  vii,  p.  735. 

41  HERMANN:  Stoffwechsel  der  Muskeln,  Hirschwald,  Berlin,  1867. 

42  TISSOT:  Archives  de  physiologic,  1894-5,  (5)  vii.  p.  469. 

43  MINOT:  Arbeiten  aus  der  physiologischen  Anstalt  zu  Leipzig,  1868,  p.  i. 

44  L.  HILL:  See  Schafer's  Text  Book  of  Physiology,  1898,  i,  p.  911. 
46  FLETCHER:  Journal  of  physiology,  1898-9,  xxiii,  p.  68. 


Carbon  Dioxide  From  Neroe  Fibres  131 

that  the  respiratory  quotient  of  the  resting  and  acting  nerve  is  nearly 
unity.  Since  he  found  that  O2  consumption  is  increased  when  stimu- 
lated, and  since  the  respiration  quotient  remains  constant  before  and 
after  the  stimulation,  he  concluded  that  it  must  give  off  more  CO2 
when  stimulated.  It  is  very  interesting  to  compare  the  O2  consump- 
tion in  this  experment  with  the  CO2  production  of  mine.46 

Taking  his  lowest  figure,  because  he  worked  in  19°  -  24°  and  I  in 
19°  -  20°,  41.7  cmm.  of  O2  amount  to  .00007  cc-  f°r  I0  milligrams  for 
ten  minutes.  My  figure  of  5.5  X  io~7  grams  for  the  same  units  may  be 
translated  to  .00027  cc.  of  CO2  (ignoring  temperature  and  pressure 

C02       00027 

correction).     Therefore——  =  -       -  =  3. 8,  the  respiratory  quotient. 

O2         00007 

As  I  have  not  determined  O2  consumption  of  the  nerve  of  Rana  pipiens, 
this  figure  has  no  particular  value,  but  the  fact  that  the  CO2  produc- 
tion is  comparatively  higher  than  02  consumption  is  a  matter  of 
considerable  interest. 

One  of  the  most  important  observations  made  by  A.  V.  Hill 47 
is  the  fact  that  he  could  not  detect  any  rise  of  temperature  in  a  frog's 
nerve  as  measured  by  an  apparatus  which  is  sensitive  to  a  change  of 
one-millionth  of  a  degree.  From  this,  according  to  his  calculation, 
he  concludes  that  not  more  than  one  single  oxygen  molecule  in  every 
cube  of  nerve  of  dimension  of  3.7  p.  can  be  used  up  by  a  single  propa- 
gated nerve  impulse.  Therefore,  he  suggested  that  an  impulse  is 
not  of  irreversible  chemical  nature  but  a  purely  physical  change. 

Although,  I  confess,  my  ignorance  makes  it  impossible  to  interpret 
his  valuable  results  from  my  observations,  I  may  add  that  these  two 
apparently  irreconcilable  facts  may  throw  light  on  the  true  nature  of 
nervous  metabolism.  Dr.  Mafliews  has  suggested  that  metabolism 
in  the  nerve  may  be  something  of  the  order  of  alcoholic  fermentation, 
which  is  not  a  direct  oxidation,  and  where  heat  production  cannot 
be  so  large  as  CO2  production,  since  the  energy  content  of  glucose  is 
only  a  trifle  higher  than  that  of  the  alcohol  produced.  The  compara- 
tively little  heat  production  in  the  case  of  working  glands  is  a  matter 
of  interest  in  this  connection.  At  any  rate  we  should  not  forget  the 

46  He  used  Rana  esculenta,  which,  by  the  way,  gives  for  the  whole  animal 
.082  g.  CO2  per  kg.  per  hour  at  17°  according  to  Schultz.     My  frog  was  Rana 
pipiens. 

47  HILL:  Journal  of  physiology,  1912,  xliii,  p.  433. 


132  Shiro  Tashiro 

anatomical  as  well  as  the  chemical  differences  between  muscle  and 
nerve.  In  this  respect  the  ratio  between  CO2  production  and  O2 
consumption  from  the  nerve  is  suggestive. 

The  extremely  small  intake  of  O2  has  another  point  of  interest  in 
relation  to  the  general  nature  of  irritability.  It  has  been  repeatedly 
reported  that  a  nerve  can  remain  excitable  several  hours  in  an  oxygen- 
free  atmosphere,  although  there  is  no  doubt  its  excitability  diminishes, 
yet  there  is  a  considerable  amount  of  evidence  to  show  that  oxygen 
is  very  closely  associated  with  the  state  of  excitability.  To  har- 
monize these  two  facts,  the  oxygen-storage  hypothesis  has  been 
suggested,  by  which  the  exhaustion  is  attributed  to  complete  consump- 
tion of  the  stored  oxygen  and  that  excitability  is  restored  when 
atmospheric  oxygen  is  readmitted.  Without  committing  ourselves  to 
this  hypothesis,  I  may  add  that  according  to  Haberlandt's  figure,  the 
resting  nerve  of  10  milligrams  will  consume  only  .0042  cc.  O2  in  ten 
hours.  If  we  take  our  figure  and  assume  that  one  volume  of  oxygen 
was  necessary  to  produce  one  volume  of  C02  (this  assumption  is  made 
without  any  significance  except  to  give  a  liberal  estimate),  the  CO2 
production  would  require  about  .015  cc.  of  02  for  ten  hours.  And  if 
we  assume  again  that  activity  will  increase  O2  consumption  in  propor- 
tion of  CO2  production,  then  it  means  that  the  nerve  when  stimulated 
takes  Up  only  .03  cc.  of  O2  during  ten  hours  stimulation.  I  am  not 
aware,  at  present,  of  the  existence  of  any  method  which  will  surely 
remove  02  as  completely  as  this  from  a  large  vessel;  and  this  is  a 
very  liberal  estimate.  My  experiences  in  rendering  the  air  free  from 
CO2  encourages  me  to  raise  the  question,  How  can  one  remove  every 
trace  of  02  from  a  nerve  fibre?  Without  having  a  correct  criterion 
for  an  oxygen-free  medium  we  cannot  at  present  consider  definitely 
any  question  of  the  relation  of  O2  to  irritability. 


CONCLUSION 

In  spite  of  all  the  negative  evidence  against  the  presence  of  meta- 
bolism in  the  nerve  fibre,  we  have  established  three  important  facts: 
namely,  (i)  A  resting  nerve  gives  off  a  definite  quantity  of  carbon 
dioxide;  (2)  stimulation  increases  CO2  production;  and  (3)  C02 
production  from"  the  resting  nerve  proportionally  decreases  as  irri- 


Carbon  Dioxide  From  Nerve  Fibres  133 

lability  diminishes.  These  facts  prove  directly  that  the  nerve  con- 
tinuously undergoes  chemical  changes,  and  that  nervous  excitability 
is  directly  connected  with  a  chemical  phenomenon.  There  is  still 
another  question  left,  namely,  Is  there  any  direct  relation  between 
excitability  and  tissue  respiration?  To  put  this  question  more  directly, 
we  may  ask:  Does  excitability  depend  on  the  respiratory  process  in 
the  protoplasm?  To  answer  these  questions  we  must  refer  to  two  facts ; 
namely  the  direct  relat  on  between  the  rate  of  respiratory  activity  and 
the  decrease  of  excitability;  secondly,  the  influence  of  reagents  on 
C02  production  and  their  effects  on  the  state  of  excitability. 

By  the  studies  of  C02  production  by  Fletcher  48  lactic  acid  forma- 
tion by  Fletcher  and  Hopkins,49  and  heat  evolution  by  A.  V.  Hill,50 
it  has  been  established  that  in  isolated  muscle,  respiratory  processes 
decrease  when  irritability  diminishes.  In  the  case  of  the  nerve, 
as  shown  in  Table  3,  C02  production  reaches  this  minimum  when 
excitability  approaches  zero.  These  relations,  however,  do  not  show 
conclusively  that  the  protoplasmic  irritability  depends  on  respiratory 
activity,  for  it  is  quite  probable  that  the  dying  nerve  may  alter  its 
physical  condition  as  well,  which  according  to  the  physical  school, 
may  consequently  alter  the  state  of  excitability. 

That  irritability  is  independent  of  the  respiratory  processes  has 
been,  hitherto  successfully  contended  in  the  case  of  the  dry  seed.  The 
works  of  Horace  Brown,  Thiselton-Dyer  51  and  others. indicate  that 
the  dry  seed  can  be  kept  alive  at  the  conditions  where  no  ordinary 
gaseous  exchanges  are  possible.  It  is  argued,  therefore,  that  life  is 
possible  without  any  metabolic  activity.52  While  a  definite  poten- 
tiality for  irritability  may  exist  without  any  metabolic  activity,  yet 
that  the  irritability  can  persist  without  respiratory  activity,  or  vice 
versa,  is  a  matter  by  no  means  settled.  In  the  case  of  ordinary 
air-dry  seed,  Waller  could  demonstrate  the  response  of  electrical 
changes  when  stimulated  although  the  detection  of  CO2  was  impossi- 

48  FLETCHER:  loc.  cit. 

49  FLETCHER  and  HOPKINS:  loc.  cit. 

50  A.  V.  HILL:  loc.  cit. 

51  THISELTON-DYER:    Proceedings  of  the  Royal  Society,  1897,  Ixii,  p.  160; 
ibid.,  Ixv,  p.  361. 

52  I  am  indebted  to  Professor  Crocker  for  his  kind  suggestion  as  to  botanical 
literature. 


134  Shiro  Tashiro 

sible.  This  failure,  however,  as  he  himself  expected,  was  due  to  the 
lack  of  delicacy  of  the  chemical  methods  for  detecting  CO2.  I  ob- 
served, with  my  apparatus  that  even  a  single  kernel  of  a  dry  seed 
gives  off  a  definite  quantity  of  CO2  as  long  as  it  is  alive.  In  ordinary 
condition  not  only  a  living  dry  seed  gives  off  more  CO2  than  the  dead 
one,  but  also  like  the  nerve,  it  always  gives  off  more  CO2  when  stimu- 
lated by  mechanical  injury.  In  the  normal  condition,  therefore,  we 
may  safely  conclude,  there  is  always  metabolic  activity  as  long  as 
the  seed  is  irritable,  and  that  in  the  different  states  of  irritability, 
the  respiratory  activity  is  proportionately  different.  At  present, 
therefore,  we  have  no  decided  evidence  which  will  prevent  us  from 
considering  excitability  as  a  function  of  respiration  under  ordinary 
conditions.  This  relation  is  more  directly  studied  by  the  use  of 
anaesthetics. 

I  have  already  demonstrated  that  an  etherized  nerve  gives  off 
considerably  less  CO2  than  the  normal.  Such  an  etherized  nerve  will 
not  give  more  C02  when  it  is  crushed.  This  may  be  interpreted 
by  some  to  mean  that  the  etherized  nerve  may  be  already  dead. 
This,  however,  is  not  the  case.  This  objection,  also,  I  have  considered 
by  studying  the  nerve  treated  with  KC1. 

•  When  the  nerve  is  treated  with  .2  m  KC1  and  then  crushed,  it  does 
not  give  an  increase  of  CO2  production.  Mathews  has  shown  that 
while  a  .2  m.  KC1  solution  renders  the  nerve  unexci table,  yet  it  will 
recover  its  excitability  by  being  replaced  into  n/8NaCl.  These  two 
facts,  therefore,  support  the  idea  that  any  agents  that  suppress  excita- 
bility of  the  nerves  also  decrease  the  CO2  production  and  that  C02 
production  by  crushing  the  nerve  must  be  largely  due  to  stimulation. 
This  hypothesis  is  strikingly  supported  by  similar  observations  on 
the  dry  seed.  Etherized  seeds  give  much  less  CO2  and  cannot  be 
stimulated  to  give  more  CO2  by  crushing,  while  under  normal  con- 
ditions, crushing  a  seed  always  increases  its  CO2  production.  Quan- 
titative experiments  in  this  direction  will  be  given  in  another  paper. 

These  facts  directly  support  Mathews'  hypothesis  that  substances 
which  suppress  irritability  must  act  on  the  tissue  respira-tion  pri- 
marily. If  such  an  hypothesis  is  correct,  we  can  easily  picture  what  is 
happening  in  the  nerve  fibre.  Vernon 53  considers  that  a  tissue 
contains  certain  substances  which  can  absorb  oxygen  from  their  sur- 
63  VERNON:  Journal  of  physiology,  1909-10,  xxxix,  p.  182. 


Carbon  Dioxide  From  Nerve  Fibres  135 

roundings  to  form  an  organic  peroxide,  and  by  the  help  of  a  peroxidase 
can  transer  this  to  amino  acid  and  carbohydrate  molecules  bound  up 
in  the  tissue,  just  as  H2  O254  can  oxidize,  with  the  help  of  an  activator, 
an  acid  of  formula  R.  CHNH2  COOH  to  CO2,  NH3  and  an  aldehyde 
RCHO,  and  then  oxidize  this  aldehyde  to  RCOOH  and  ultimately  to 
CO2  and  H2  O.  Poisons  such  as  HNC,  NaHSO3  and  NaF,  which  he 
found  to  decrease  CO2  production,  temporarily  paralyzed  respiration, 
he  thought,  by  uniting  with  aldehyde  groups,  while  formaldehyde,  acid 
and  alkali  temporarily  paralyze  CO2  forming  power  of  the  tissue  by 
destroying  the  peroxidase.  The  organic  peroxide,  though  it  can  still 
affect  some  oxidation,  cannot  of  itself  carry  it  to  the  final  CO2  stage. 
Recovery  of  C02  forming  power  is  due  to  the  regeneration  of  the 
peroxidase. 

Although  I  doubt  that  such  a  process  occurs  in  nervous  respiration, 
the  idea  of  two  similar  metabolic  phenomena  involved  in  the  nervous 
metabolism  is  very  helpful  to  understand  the  behavior  of  the  nerve 
during  continued  activity.  Most  recently  Tait  discovered  that  a 
refractory  period  has  two  phases,  absolute  and  relative.55  When 
he  treated  the  sciatic  nerve  of  a  frog  with  yohimbine,  the  relative 
phase  is  greatly  prolonged,  while  the  absolute  one  is  little  affected, 
a  result  quite  different  from  other  common  anaesthetics.  Waller56 
has  already  observed  that  protoveratrin  slows  up  the  positive 
variation  of  the  nerve,  while  the  negative  variation  is  little  in- 
fluenced. Waller  contends  that  this  drug  does  not  alter  cata- 
bolic  change,  but  retards  anabolic  activity  to  a  considerable 
degree.  Since  pharmocological  action  on  animals  of  protoveratrin 
and  yohimbine  are  very  similar,  Tait  concludes  that  these  drugs 
must  attack  the  nerve  in  similar  manner,  and  that  a  refractory  period, 
too,  must  consist  of  two  phases  corresponding  to  the  catabolic  and 
anabolic  processes  which  Waller  observed  in  the  case  of  protovera- 
trinized  nerves.  Thus,  he  considers  that  his  "  absolute  phase"  of 
the  refractory  period  corresponds  to  negative  variation  or  catabolic 
process  of  the  nerve,  and  the  "  relative  "  to  the  positive  return  or 
anabolic.  Yohimbine,  in  other  words,  retards  anabolic  processes  con- 
siderably, thus  prolonging  the  refractory  period,  or  increasing  nerve 

54  DAKIN:  Journal  of  biological  chemistry,  1908,  iv,  pp.  63,  77,  81,  227. 

55  TAIT:  Journal  of  physiology,  1912,  xl,  p.  xxxviii. 

56  WALLER:  Brain,  1900,  xxiii,  p.  21. 


136  Shiro  Tashiro 

fatigue  easily.  These  considerations  suggest  very  strongly  that  the 
absence  of  fatigability  in  the  nerve  as  measured  by  the  ordinary 
methods,  is  not  a  question  of  absence  of  metabolism,  but  merely  the 
speed  by  which  these  two  processes  come  to  an  equilibrium. 

Although  we  have  an  infinite  number  of  facts  still  unexplainable, 
by  our  present  knowledge  of  nerve  physiology,  we  have  established  a 
few  new  facts  around  which  we  may  build  up  some  idea  concerning 
this  most  essential  phenomena  of  living  matter,  —  i.e.,  irritability. 
As  to  the  true  nature  of  the  nerve  impulse,  I  can  only  confess  my 
ignorance. 

SUMMARY 

1.  All  nerve  fibres  give  off  C02.     The  resting,  isolated  nerve  of 
the  spider  crab  produces  6.7  X  io~7  gram  per  10  milligrams  per  ten 
minutes.     The  frog's  sciatic  5.5  X  io~7  grams. 

2.  When  nerves  are  stimulated  they  give  off  more  CO2.     The 
nerve  of  the  spider  crab  claw  produces  16.  X  icr7  gram  when  stimu- 
lated, the  frog  nerve  14.2  X  icr7  grams.     The  rate  of  increase  of 
CC>2  by  stimulation  amounts  to  about  2.5  times. 

3.  The  C02  output  of  resting  nerve  is  due  to  a  vital  active  process. 

4.  Anaesthetics  greatly  reduce  the  carbon  dioxide  output  of  nerves 
and  dry  seeds. 

5.  Mechanical,  thermal  and  chemical  stimulation  also  increases 
the  carbon  dioxide  output  of  nerves. 

6.  Single  dry  living  seeds  (oat,  wheat,  etc.)  react  in  most  par- 
ticulars similar   to   nerves  as   regards   their   irritability,   relation  to 
anaesthetics,  mechanical  stimulation  and  carbon  dioxide  outputs. 

7.  The  general  conclusion  is  drawn  that  irritability  is  directly 
dependent  upon  and  connected  with  tissue  respiration  and  is  primarily 
a  chemical  process.     These  results  strongly  support  the  conception 
that  conduction  is  of  the  nature  of  a  propagated  chemical  change. 

To  Prof.  A.  P.  Mathews,  under  whose  direction  I  have  carried  on 
these  experiments,  I  express  my  appreciation  and  gratitude.  For 
many  suggestions,  I  am  under  obligation  to  Dr.  F.  C.  Koch. 


Reprinted  from  the  American  Journal  of  Physiology 
Vol.  XXXII  — June  2,  1913  — No.  II 


A  NEW  METHOD  AND  APPARATUS  FOR  THE 

ESTIMATION  OF  EXCEEDINGLY  MINUTE 

QUANTITIES   OF   CARBON  DIOXIDE1 

BY  SHIRO  TASHIRO 

[From  the  Department  of  Biochemistry  and  Pharmacology,  the  University  of  Chicago,  and  the 
Marine  Biological  Laboratory,  Woods  Hole,  Mass.] 

IN  connection  with  the  study  of  the  metabolism  of  the  nerve  fibre, 
I  undertook,  at  the  suggestion  of  Prof.  A.  P.  Mathews,  to  work 
out  a  method  for  the  detection  of  exceedingly  minute  quantities  of 
carbon  dioxide.  Following  a  suggestion  made  by  Dr.  H.  N.  McCoy, 
a  very  simple  method  was  devised,  which  I  reported  first  to  the 
Chicago  Section  of  the  American  Chemical  Society; 2  later  in  con- 
junction with  Dr.  McCoy,  its  further  details  were  reported  to  the 
Analytic  Section,3  of  the  Eighth  International  Congress  of  Applied 
Chemistry.  The  principle  of  the  new  method  is  as  follows : 

1 .  Exceedingly  minute  quantities  of  carbon  dioxide  can  be  precipi- 
tated as  barium  carbonate  on  the  surface  of  a  small  drop  of  barium 
hydroxide  solution. 

2.  When  a  drop  of  barium  hydroxide  is  exposed  to  any  sample  of 
gas  free  from  carbon  dioxide,  it  remains  perfectly  clear,  but  when 
more  than  a  quite  definite  minimum  amount  of  carbon  dioxide  is  intro- 
duced, a  precipitate  of  carbonate  appears,  detectable  with  a  lens. 

3.  By  determining,  therefore,  the  minimum  volume  of  any  given 
sample  of  a  gas  necessary  to  give  the  first  visible  formation  of  the 
precipitate,  its  carbon  dioxide  content  can  be  estimated  accurately, 
since  this  volume  must  contain  just  the  known  detectable  amount  of 
carbon  dioxide. 

1  One  of  these  apparati  was  described  at    the  biochemical  section,  Eighth 
International  Congress  of  applied  chemistry,  September,  1912;  see  also,  Journal 
of  biochemistry,  1913,  xiv,  p.  xli. 

2  May  18,  1912. 

3  Original  Communication:  Eighth  International  Congress  of  applied  chemis- 
try, 1912,  i,  p.  361. 


138 


Shiro  Tashiro 


I  have  constructed  two  apparati,  based  on  this  principle,  which 
are  especially  adapted  for  the  estimation  of  the  output  of  carbon  diox- 
ide for  very  small  biological  specimens.  With  these  apparati,  one 
cannot  only  detect  easily  a  very  small  amount  of  gas,  given  off  by  a 
small  dry  seed,  or  a  small  piece  of  a  frog's  sciatic  nerve,  but  can  also 
estimate  it  with  considerable  accuracy. 

The  apparatus  shown  in  Fig.  i 
consists  of  two  glass  bulbs.  The 
upper  bulb  A,  is  a  respiratory 
chamber,  having  a  capacity  of  about 
15  c.c.,  which  can  be  diminished  to 
9  c.c.  by  means  of  mercury.  The 
lower  bulb  B  is  an  analytic  chamber 
with  a  volume  of  25  c.c.,  which  can 
be  made  to  5  c.c.  by  filling  up  with 
mercury.  These  two  bulbs  are  con- 
nected with  a  capillary  stop-cock  D. 
The  respiratory  chamber  is  fitted 
with  a  tight  glass  stopper,  R,  which 
is  connected  to  a  three-way  capillary 
stop-cock  C.  This  glass  stopper  is 
so  arranged  that  the  chamber  can 
be  sealed  by  putting  mercury  above 
the  stopper. 

The  tubes  are  thick  walled  capillaries  of  about  i  mm.  internal 
diameter,  excepting  upturned  tubes  inside  the  bulbs,  which  should 
be  rather  thin  walled,  especially  at  F  and  H,  where  it  is  widened  to  an 
internal  diameter  of  about  2  mm.  It  is  important  that  the  glass  of 
which  these  tubes  are  made  should  be  of  a  quality  not  readily  attacked 
by  barium  hydroxide. 

The  details  of  the  method  of  procedure  are  as  follows : 

The   apparatus   is   first   cleaned   and   dried.4    The   specimen   is 

4  The  apparatus  is  made  in  such  a  way  that  it  can  be  cleaned  and  dried  in 
ten  minute's  without  being  taken  apart.  For  this,  the  stop-cock  D  is  closed  and 
E  and  L  are  opened.  The  arm  at  L  is  connected  to  the  suction  pump.  Then 
a  little  acidulated  water  is  introduced  through  G.  By  closing  E,  and  opening  D 
and  G,  the  excess  of  water  is  drained  off.  Then  the  process  is  repeated  with  dis- 
tilled water,  alcohol,  and  alcohol  ether.  The  last  drying  is  completed  by  passing 
a  current  of  air  through  G  while  D  is  closed. 


FIGURE  1 
One- third  the  actual  size. 


Apparatus  For  Estimating  Carbon  Dioxide  139 

placed  on  a  glass  plate  5  and  weighed.  The  glass  plate  is  hung  on 
n  and  m,  which  are  electrodes  fused  into  the  side  of  the  respiratory 
chamber  A.  The  chamber  is  now  closed  with  the  stopper  R  and 
sealed  with  mercury.  Through  L,  a  connection  is  made  with  a 
pump  6  and  about  20  c.c.  of  mercury  is  introduced  through  G.  Not 
too  much  mercury  should  be  used;  its  surface  should  not  be  within 
5  mm.  of  the  cup  F.  Then  wash  the  whole  apparatus  with  carbon 
dioxide-free  air,7  which  is  introduced  through  C,  by  successive 
evacuations.  After  the  evacuation  and  washing  out  with  pure  air, 
which  is  repeated  three  or  four  times,  the  pressure  inside  of  the  bulbs 
is  made  equal  to  the  atmospheric  pressure  by  adjusting  it  at  the  nitro- 
meter in  the  usual  fashion.  Stop-cock  E  is  then  closed,  and  the 
space  between  E  and  L  is  evacuated  so  that  the  barium  hydroxide 
can  rush  in,  a  process  which  is  very  advantageous  to  obtain  a  clear 
barium  hydroxide  solution.  Then  clear  barium  hydroxide  solution 
is  run  in  through  L.  By  opening  E  very  slowly  and  carefully,  the  solu- 
tion is  now  introduced  into  the  chamber  so  that  a  small  drop  stands 
up  upon  the  upturned  end  of  the  capillary  at  F.  Then  the  connection 
between  the  two  chambers  is  closed  by  D.  It  is  imperative  that  this 
drop  of  the  solution  should  be  perfectly  clear  at  the  start.  If  no 
deposit  of  barium  carbonate  forms  on  the  surface  of  the  drop  within 
ten  minutes,8  a  portion  of  the  sample  gas  is  drawn  into  B  by  with- 
drawing mercury  through  G  and  opening  the  stop-cock  D.  The 
volume  of  mercury  withdrawn,  which  may  be  readily  determined  by 
volume,  or  more  accurately  by  weight,  gives  the  volume  of  the  sample 

5  The  kind  of  glass  plate  used  in  connection  with  the  nerve  and  small  animals 
like  Planaria  is  shown  on  p.  120,  Fig.  i.     (The  first  paper.) 

6  The  pump  should  be  capable  of  giving  a  vacuum  of  at  least  25  or  30  mm.  of 
mercury. 

7  Air  cannot  be  freed  completely  from  carbon  dioxide  by  passing  it  through 
wash  bottles.     In  my  work,  carbon  dioxide-free  air  is  prepared  by  shaking  air  with 
twenty  per  cent  solution  of  sodium  hydroxide  in  a  tightly-stoppered  carboy,  fitted 
with  suitable  tubes.     When  this  is  to  be  used,  it  is  driven  into  a  nitrometer  which 
is  filled  with  less  concentrated  alkaline  solution  (a  weak  solution  is  used  so  that 
the  chamber  may  not  be  too  dry)  by  displacing  it  by  running  in  a  solution  of 
sodium  hydroxide.     After  each  evacuation,  this  air  is  introduced  from  the  nitro- 
meter into  the  chamber  A  through  stop-cock  C. 

8  If  no  precipitate  appears  within  ten  minutes,  it  is  a  sure  control  that  the 
apparatus  is  free  from  carbon  dioxide. 


140  Shiro  Tashiro 

gas  taken  from  the  respiratory  chamber,  since  the  pressure  in  A  and 
B  is  kept  equal  to  the  atmospheric  during  the  transfer. 

One  now  watches  the  surface  of  the  drop  at  F  with  a  lens  to  see 
whether  any  formation  of  barium  carbonate  occurs  within  ten  minutes. 
With  this  apparatus,  I  have  repeatedly  introduced  accurately  known 
quantities  of  carbon  dioxide  of  very  high  dilution  into  B  in  the  manner 
just  described  and  as  a  result  have  found,  with  remarkable  regularity, 
that  i.o  X  io~7  gram  of  carbon  dioxide  is  the  minimum  amount 
which  will  cause  a  formation  of  barium  carbonate  within  a  period  of 
ten  minutes.  Smaller  amounts  of  carbon  dioxide  give  no  visible 
results ;  while  larger  amounts  give  a  deposit  more  rapidly,  and  appear 
in  larger  quantities.  This  minimum  detectable  amount  i.o  X  io-7 
gram  is  about  the  amount  which  is  contained  in  J  c.c.  of  natural  air, 
in  which  we  assume  3.0  parts  of  carbon  dioxide  in  10,000  by  volume.9 

In  order  to  determine  the  concentration  of  carbon  dioxide  in  the 
respiratory  chamber,  one  must  first  find,  for  the  apparatus  used,  the 
minimum  detectable  amount  of  carbon  dioxide.  Then  one  finds,  by 
trial,10  the  minimum  volume  of  gas  necessary  to  give  the  first  visible 
formation  of  barium  carbonate.  This  volume  must,  therefore,  con- 
tain the  known  minimum  detectable  amount  of  carbon  dioxide.  From 
the  ratio  between  this  volume  and  the  original  volume  of  the  respira- 
tory chamber,  out  of  which  this  amount  is  withdrawn,  the  absolute 

9  LETTS  and  BLAKE:  Proceedings  of  the  Royal  Dublin  Society,  1899-03,  ix, 
p.  107. 

10  In  the  case  of  biological  problems,  when  the  specimen  gives  off  carbon 
dioxide  continuously,  and  sometimes  at  different  rates,  varying  with  the  time,  it 
is  much  simpler  not  to  attempt  to  determine  the  minimum  volume  by  a  continuous 
trial  with  the  same  sample;  but  instead  to  repeat  the  experiments  with  a  series 
of  samples  of  known  weights  for  a  known  time,  and  determine  the  minimum 
volumes  which  give  the  precipitates,  and  the  maximum  volumes  which  do  not 
give  the  precipitates.    In  this  way,  it  can  easily  be  calculated  what  is  the  mini- 
mum volume  which  gives  the  precipitate  for  the  given  weight  of  the  specimen  for 
a  given  time.    Table  I  on  page  1 14  will  illustrate  this  more  clearly. 

Another  upturned  cup  H  provided  in  the  respiratory  chamber  A  is  used  in 
case  only  the  qualitative  detection  of  C02  is  wanted.  In  such  a  case,  the  perfectly 
clear  barium  hydroxide  solution  is  introduced,  after  the  necessary  cleaning  and 
washing,  to  the  respiratory  chamber,  forming  the  usual  drop  at  H  instead  of  F. 
It  should  be  noted  that  in  case  a  smaller  capacity  is  necessary  for  the  respiratory 
chamber,  the  mercury  is  introduced  by  a  pipette  to  the  bottom  of  the  chamber 
at  K. 


Apparatus  For  Estimating  Carbon  Dioxide 


141 


quantity    of    carbon    dioxide,    given    by    the   specimen,    may    be 
computed. 

At  the  suggestion  of  Dr.  F.  C.  Koch,  another  apparatus  was  con- 
structed, which  provides  a  control  drop  of  the  barium  hydroxide 
solution,  side  by  side  with  the  other.  The  apparatus  (Biometer)  shown 
in  Fig.  2,  although  it  appears  complex,  is  nothing  more  than  apparatus 
i,  inclined  90°,  but  each  of  its  chambers  is  provided  with  a  barium 
hydroxide  cup  d  and  f .  It  is  made  of  glass  consisting  of  two  respi- 
ratory chambers,  serving  also  as  analytic  chambers,  connected  by  a 
three-way  stop-cock  L,  the  other  arm  of  which  is  connected  to  one 
arm  of  another  three-way  stop-cock  K.  Each  of  the  other  two  arms 
of  stop-cock  K  is  connected  to  a  nitrometer  W  and  X.  The  nitro- 


FIGURE  2.     Biometer,  one-third  actual  size. 

The  shaded  portions  of  the  apparatus  indicate  the  rubber  connection  which  is  first 
coated  by  shellac,  and  then  sealed  with  a  special  sealing  wax.  Some  parts  are 
also  sealed  with  mercury. 

meter  on  the  right,  is  connected  to  a  carboy  with  air  free  of  COz',  and 
the  other,  on  the  left,  to  a  similar  reservoir  with  air  free  of  CO2  plus 
any  gas  which  is  desired  as  a  medium  for  conducting  the  experi- 
ment. Chamber  A  is  drawn  to  a  capillary  stop-cock  C;  chamber  B 
is  drawn  to  the  three-way  stop-cock  G,  one  arm  of  which  is  con- 
nected with  a  mercury  burette  T,  which  is  used  for  adjusting  the 
pressure.  Both  of  the  chambers  have  a  capacity  of  20  to  25  c.c. 
and  are  provided  with  a  pair  of  platinum  electrodes  n  and  m,  and 
also  with  the  glass  stoppers  S  and  R,  which  can  be  sealed  as  usu*al 
with  mercury.  The  pump  is  connected  through  J,  and  the  barium 


142  Shiro  Tashiro 

hydroxide  solution  is  introduced  through  V  to  d  and  f,  where  drops 
are  formed  as  before. 

As  stated  above,  this  apparatus  can  be  used  for  the  combined 
purposes  of  qualitative  detection,  quantitative  estimation,  and  com- 
parative determination  of  the  output  of  CO2  from  the  various  biolog- 
ical specimens.  It  has  a  decided  advantage  over  the  other  in  the 
fact  that  we  have  a  control  drop,  side  by  side,  under  exactly  the  same 
conditions,  and  that  the  comparative  estimation  of  CO2  produced  by 
different  specimens  can  be  made  very  easily  and  accurately.  The  de- 
tailed method  of  procedure  is  described  under  three  different  headings : 

(a)  For  the   Qualitative  Detection  of  Carbon  Dioxide.  —  After 
the  apparatus  is  cleaned  and  dried,11  a  weighed  tissue  is  placed  on  the 
glass  plate  and  hung  on  n  and  m  of  the  chamber  A,  and  no  tissue  in 
the  other  chamber.     After  both  chambers  are  closed  with  the  stop- 
pers S  and  R  and  sealed  with  mercury,  they  are  so  filled  with  mercury 
that  the  remaining  volumes  in  both  chambers  are  now  exactly  the 
same.     The  chambers  are  now  evacuated  and  washed  with  pure  air. 
When  evacuation  and  washing  with  pure  air  is  complete,  the  pressure 
is  made  atmospheric,  by  adjusting  with  the  nitrometer  the  connec- 
tion between  A  and  B  is  then  closed  with  stopcock  L.     If  any  CC>2 
is  given  off  by  the  tissue,  the  desposit  of  carbonate  will  soon  appear 
on  d,  while  in  the  control  chamber  the  drop  on  f  remains  perfectly 
clear.     In  order  to  avoid  any  possible  error  of  a  technical  nature  this 
experiment  is    repeated  by   exchanging   the    chambers,    now   using 
chamber   B   for  the  respiratory  chamber    and    the   other   A   as   a 
control. 

(b)  For   Comparative   Estimation   of   CO2  from   Two   Different 
Samples.  —  By   repeated   quantitative   experiments,   it   was   found 
that  the  speed  with  which  the  first  precipitate  appears  and  the  sizes 
of  the  deposits  on  the  drops  at  d  and  f  represent  corresponding  quanti- 
ties of  carbon  dioxide.     Thus  with  remarkably  simple  means,  we  can 
determine  simultaneously  the  comparative  outputs  of  the  gas  from  two 
different  tissues  or  from  the  same  tissues  under  different  conditions. 
The  method  of  procedure  is  best  illustrated  by  the  following  example. 
Two  pieces  of  the  sciatic  nerve  are  isolated  from  the  same  frog  and 
exactly  weighed.     One  piece  is  laid  on  one  glass  plate,  and  the  other 

11  This,  too,  can  be  cleaned  and  dried  without  being  taken  apart.  See  foot- 
note on  p.  138. 


Apparatus  For  Estimating  Carbon  Dioxide  143 

on  the  other  plate  in  such  a  way  that  one  part  of  the  nerve  lies  across 
the  electrodes  of  the  glass  plates  as  shown  in  Fig.  i,  page  120.  In 
this  way,  when  the  plates  are  hung  on  the  electrodes  n  and  m,  any 
desired  nerve  can  be  stimulated  with  the  induction  current.  These 
plates  are  now  hung  on  the  electrodes  in  each  chamber,  and  the  usual 
procedure  is  followed  for  the  cleaning  and  the  washing  of  the  appara- 
tus to  make  it  C02  free.  After  the  connection  between  the  two 
chambers  is  closed  by  means  of  stop-cock  L,  the  nerve  in  chamber 
A  is  stimulated  by  the  current.  Then  if  one  can  watch  over  the 
surfaces  of  the  drops  carefully  from  the  start,  he  finds  the  first  deposit 
of  the  carbonate  on  cup  d  of  chamber  A  in  which  the  stimulated  nerve 
is  placed.  Later,  the  total  amount  of  the  precipitates  grows  much 
larger  in  the  case  of  this  cup.  This  increased  output  of  the  carbon 
dioxide  from  the  stimulated  nerve,  thus  observed,  can  be  duplicated 
by  repeating  the  similar  experiment,  after  exchanging  the  chambers, 
as  usual.  This  comparative  estimation  can  be  more  accurately  made 
by  exact  quantitative  measurement,  the  method  for  which  the  follow- 
ing will  illustrate. 

(c)  For  Quantitative  Measurement  of  Gas.  — •  The  detailed 
method  is  exactly  analogous  to  that  of  apparatus  i.  Here  we  use 
chamber  B  as  the  respiratory  chamber  and  A  as  the  analytic  cham- 
ber. Barium  hydroxide  should  be  introduced  into  chamber  A  only 
at  d,  and  the  stop-cock  F  is  always  closed  except  at  the  time  of  wash- 
ing. The  pressure  should  be  adjusted  by  mercury  burette  T,  or  by 
the  potash  bulb  of  the  nitrometer.  In  case  the  mercury  burette  is 
used,  the  remaining  volume  in  the  respiratory  chamber  should  be 
recorded.12  The  introduction  of  a  known  amount  of  gas  from  the 
respiratory  chamber  B  to  the  analytic  chamber  A  is  accomplished 
by  withdrawing  the  mercury  from  C  into  a  very  narrow  graduated 
cylinder,  while  the  stop-cocks  L  G  and  H  are  opened.  After  a  quick 
adjustment  of  the  mercury  burette  to  equalize  the  pressure,  the  stop- 
cock L  is  closed  and  the  presence  of  carbonate  is  looked  for  exactly 
in  the  same  manner  as  described  in  connection  with  the  other  appara- 
tus, determining  the  minimum  volume  that  gives  the  precipitate  for 
the  known  mass  of  tissue  for  a  known  time. 

12  The  bulbs  are  marked  at  the  point  where  their  capacity  became  15  c.  c. 
by  introducing  mercury.  The  variation  of  capacity  can  easily  be  read  by  noting 
the  mercury  burette. 


144  Shiro  Tashiro 

In  summarizing,  I  may  emphasize  the  following  points: 

1.  Particular  care  must  be  taken  to  test  the  air- tightness  of  the 
apparatus. 

2.  Purifying  the  air  must  be  done  with  greatest  care,  as  this  is 
essential. 

3.  The  apparatus  must  be  perfectly  dry. 

4.  A  weak  suction  pump  cannot  be  compensated  by  frequency  of 
washing. 

5.  As  long  as  the  ratio  between  the  c.c.  taken  from  the  chamber 
and  the  original  volume    of   the  chamber   is   needed/  it  is    most 
important  to  have  the  pressure  in  A  and  B  equal  to  the  atmos- 
pheric.    If  this  is  accomplished  we  can  neglect  any  caution  against 
pressure  and  temperature  variations  —  a  correction  which  is  always 
necessary  for  ordinary  methods  of  analysis  of  exceedingly  minute 
quantities  of  any  gas. 

In  devising  this  method  and  in  constructing  this  apparati,  I  am 
under  great  obligation  to  Professors  McCoy  and  A.  P.  Mathews  and 
to  Dr.  F.  C.  Koch. 

In  order  to  test  the  accuracy  with  which  an  estimate  of  concen- 
tration of  carbon  dioxide  could  be  made,  many  determinations  were 
carried  out  with  samples  of  air  which  contained  accurately  known 
concentrations  of  carbon  dioxide  prepared  by  Dr.  F.  C.  Koch.  The 
experimenter  did  not  learn  the  concentrations  of  the  samples  until 
after  the  analysis  had  been  completed.  In  making  up  the  test  sam- 
ples, pure  carbon  dioxide,  made  by  heating  sodium  bicarbonate  was 
diluted  with  the  carbon  dioxide  free  air  several  times  in  succession, 
as  illustrated  by  the  following  example:  5.5  c.c.  of  pure  carbon  diox- 
ide was  diluted  to  52.0  c.c.  over  mercury  and  thoroughly  mixed; 
5.5  c.c.  of  the  first  mixture  was  diluted  to  52.0  c.c.;  i.i  c.c.  of 
the  second  was  diluted  to  50.7  c.c.;  of  this  third  mixture  5.6  c.c. 
was  received  from  Dr.  Koch.  I  diluted  this  a  fourth  time  to 
255.6  c.c.  to  form  a  mixture  to  be  analyzed.  The  following  observa- 
tions were  made:  0.5  c.c.  was  introduced  into  the  apparatus  and  pro- 
duced no  precipitate  in  ten  minutes;  0.5  c.c.  more  of  the  same  sample, 
gave  no  precipitation  in  another  interval  of  ten  minutes;  0.5  c.c. more, 
a  total  of  1.5  c.c.,  was  run  into  the  bulb.  In  six  minutes  the  first 
evidence  of  a  precipitate  appeared  on  the  surface  of  the  drop  at 
d  of  apparatus  2  and  in  eight  minutes  was  well  developed.  Since 


Apparatus  For  Estimating  Carbon  Dioxide 


145 


the  amount  of  carbon  dioxide  required  to  give  the  precipitate  is  i.o 
X  io~7  grams,  this  amount  is  contained  in  1.5  c.c.  of  the  sample 
or  i  c.c.  contained  6.7  X  io~8  grams  of  carbon  dioxide.  The 
amount  of  carbon  dioxide  actually  contained  in  the  sample  was 
5.5  X  5-5  X  7-1  X  5-6 


52  X  52  X  50-7  X  255.6 


c.c.  =  6.2  X  io-8  grams. 


In  six  such  determinations,  all  made  with  samples  the  concentra- 
tion of  which  were  unknown  to  the  experimenter  at  the  time  of  the 
analysis,  the  results  given  in  the  following  table  were  obtained: 


Volume  of  sample  re- 
quired to  give  a 
precipitate 

Weight  of  carbon  dioxide  in  one  c.c. 

Found 

Taken 

1.0    c.c. 

1.0    X  IO-7  g. 

0.92  X  IO-7  g. 

.5    c.c. 

2.      X  IO-7  g. 

2.3    X  IO-7  g. 

.55  c.c. 

1.82  X  IO-7  g. 

1.83  X  IO-7  g. 

1.5    c.c. 

.67  X  IO-7  g. 

0.62  X  IO-7  g. 

2.25  c.c. 

.45  X  IO-7  g. 

0.45  X  IO-7  g. 

ERRATA 

IN  JUNE  NUMBER  OF  THE  AMERICAN  JOURNAL  OF  PHYSIOLOGY 
(VOL.  XXXII,  No.  II) 

Substitute  " apparatus"  for  "apparati"  in  the  following  places: 

Page  no,  lines  7,  n,  23. 
Page  129,  line  i. 
Page  137,  line  28. 
Page  144,  line  16. 

Substitute  "7.1  cc."  for  "  i.i  cc."  on  page  144,  line  29. 

In  figure  i ,  page  1 20,  correct  as  indicated  in  the  following  drawing 


THE  LIBRARY  . 

UNIVERSITY  OF  CALIFORNIA ///)U/ 

San  Francisco  *f  ' 

THIS  BOOK  IS  DUE  ON  THE  LAST  DATE  STAMPED  BELOW 

Books  not  returned  on  time  are  subject  to  fines  according  to  the  Library 
Lending  Code.  A  renewal  may  be  made  on  certain  materials.  For  details 
consult  Lending  Code. 


14  DAY 

JAN  2  2 


RETURNEE 
APR  -  3  1979 
14  DAY 
JUL271994 

14  DAY 

AUG  1  5  1994 


RETURNED 

SEP  2  8  19! 


Series  4128 


618604 


3   1378  00618  6046 


o^  -*--  8689 

ciTrbon'dioxicie  produc- 
ion  from  nerlve  fibres-. 


